Photoactive Compounds and Methods for Biomolecule Detection and Sequencing

ABSTRACT

Disclosed herein are compositions, probes, devices, and processes useful for detecting specific reactions and binding interactions with biological molecules. In certain embodiments, methods of binding one or more biomolecules to a solid support are disclosed. Methods of generating site-specific sequences for one or more biomolecules from a solid support are also disclosed. Biological complexes generated by these methods are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/991,706, filed on May 29, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/511,786, filed May 26, 2017, eachincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 27, 2018, isnamed 40697US_CFR_sequencelisting.txt and is 6,997 bytes in size.

BACKGROUND

Microarray technologies can facilitate detection of many features persquare centimeter. This can include detection via probe bindingmethodologies to detect or accurately quantify the presence ofbiomolecules and to characterize these biomolecules, e.g., bydetermining a specific conformation or sequence. As more informationcontinues to be processed at faster rates, certain features start tobecome problematic as limiting to the amount of information that can beobtained. For example, many detection technologies, such as probedetection and sequencing rely on monitoring fluorophores anddistinguishing fluorophores bound to known probes. The use offluorophore tags limits the size of the features on a chip due to thediffraction limit, and also can be difficult to detect at smallconcentrations. Alternative detection technologies exist, but they needfurther development to provide a suitable improvement tofluorophore-based detection technologies. Therefore, what are needed arealternatives to fluorophore-based detection technologies to improvedetection accuracy and facilitate a reduction of feature size for higherthroughput and more efficient detection.

As one example, a typical solid support-based detection assay isgenerally comprised of probes that bind to biologically relevant oractive molecules for example, RNA, DNA, or peptides. Probes that bind totarget molecules or the target molecules themselves can be covalentlyattached to a solid planar surface for example, glass, polymer (bead oreven plastic composites), or most often, a silicon chip. Additionally,instruments are needed to handle samples (automated robotics), to readthe reporter molecules (scanners) and analyze the data (bioinformatictools). Recently, science has moved toward a unitary machine to performthese much need analyses. In order to marry the chemistry and biologywith electronics, silicon wafers are most often used as the solidsupport or substrate. The term “lab on a chip” has since been coined todescribe such an arrangement.

Microarrays technology can facilitate monitoring of many probes persquare centimeter. The advantages of using multiple probes include, butare not limited to, speed, adaptability, comprehensiveness and therelatively cheaper cost of high volume manufacturing. The uses of suchan array include, but are not limited to, diagnostic microbiology,including the detection and identification of pathogens, investigationof anti-microbial resistance, epidemiological strain typing,investigation of oncogenes, analysis of microbial infections using hostgenomic expression, and polymorphism profiles.

Recent advances in genomics have culminated in sequencing of entiregenomes of several organisms, including humans. Genomics alone, however,cannot provide a complete understanding of cellular processes that areinvolved in disease, development, and other biological phenomena;because such processes are often directly mediated by polypeptides.Given that huge numbers of polypeptides are encoded by the genome of anorganism, the development of high throughput technologies for analyzingpolypeptides, amongst many other diverse biomolecules, is of paramountimportance.

Peptide arrays with distinct analyte-detecting regions or probes can beassembled on a single substrate by techniques well known to one skilledin the art. A variety of methods are available for creating a peptidemicroarray. These methods include: (a) chemo selective immobilizationmethods; and (b) in situ parallel synthesis methods which can be furtherdivided into (1) SPOT synthesis and (2) photolithographic synthesis.These methods are labor intensive and not suited for high throughput.These peptide arrays are expensive to manufacture, have lowrepeatability, may be unstable, require stringent storage conditions,take a long time to manufacture, and are limited in other ways. Further,while peptide-nucleic acid arrays are useful for identifyingbiomolecules, there is currently no way to deduce the binding strengthor sequence.

What is needed therefore, are improved substrates or arrays and methodsto elucidate and replicate biomolecule sequences and measure the bindingof one or more biomolecules.

As another specific example, next generation sequencing technologies,including sequencing-by-synthesis, continue to pursue the goal ofproviding rapid sequencing data at a reasonable cost. This can be usedto provide improved health care through individualized medicine andimproved diagnostics. Despite many improvements in the past decades,this technology still has limitations in cost and throughput thatprevent widespread use. Overcoming these limitations can provide adramatic impact in several fields, including comparative genomics,high-throughput polymorphism detection, mutation screening,metagenomics, and transcriptome profiling.

Sequencing by synthesis of template DNA bound to a surface is commonlydone using fluorophore-labeled, reversible terminator nucleotides. Thesenucleotides generate a signal corresponding to the sequence of asurface-bound template strand when incorporated into a complementarygrowing strand. For example, U.S. Pat. No. 7,622,279 teaches afluorescence-based method for sequencing four modified nucleotides withphoto-cleavable fluorescence molecules bound to the side chain of thefour nucleic acid bases.

However, optical detection methods have a limited minimum feature sizedue to diffraction limited detection of fluorophores. Furthermore,imaging of an array of signals and processing the image to generatediscrete endpoints can take time and be computationally demanding. Thus,alternative methods of nucleotide identity detection, such as electronicdetection are also being explored.

One such method of electronic detection, Ion Sensitive Field EffectTransistors (ISFET), is able to detect small changes in the pH of areaction volume. Non-optical genome sequencing using ISFET has beenperformed by adding a single nucleotide at a time to detect the releaseof an H+ ion upon incorporation of a correct base pair by a polymeraseinto a growing strand. However, this method is limited by therequirement of separate sequential addition of four individualnucleotides to determine the identity of the next nucleotide. UsingISFET detection, samples can be distributed on an array at thesub-micron level, and multiple arrays can be read simultaneously in asingle device.

What is needed therefore, are improved methods, compositions, substratesand arrays for determining a polynucleotide sequence based on electronicdetection to allow reduce feature size on an array for increasedinformation density with output that allows for more efficient analysis.

Furthermore, arrays comprising primers or probes to bind to targetsequences to allow sequencing are also needed to enable efficientbinding of target polynucleotides for to an array for subsequentsequencing. Also needed are methods and compositions for manufacturingarrays comprising the probes.

SUMMARY

According to some embodiments, provided herein is a probe capable ofbinding specifically to a target biomolecule, wherein said probe isbound to a photoactive group. In some embodiments, the photoactive groupis a photoacid generator or a photobase generator.

In some embodiments, the photobase generator produces an organiccompound having a pKa of 9 or higher, 10 or higher, 11 or higher, 12 orhigher, 13 or higher, or 14 or higher upon exposure to an activatingradiation. In some embodiments, the photoacid generator produces anorganic compound having a pKa of 5 or lower, 4 or lower, 3 or lower, 2or lower, or 1 or lower upon exposure to an activating radiation.

In some embodiments, the photoacid generator is selected from the groupconsisting of: an o-acyloxime, a benzoyloxycarbonyl derivative, aphotoactive carbamates, an oxime ester compounds, an ammonium compound,a benzoin compound, a dimethoxybenzyl urethane compound, anorthonitrobenzyl urethane compound, an aromatic sulfonamide, analpha-lactams, and an N-(2-arylethenyl) amide. In some embodiments, thephotoacid generator is selected from: the photoactive group of PM1 andthe photoactive group of PM2.

In some embodiments, the photobase generator is selected from the groupconsisting of: a 2-hydroxy-2-phenylacetophenone N-cyclohexyl carbamate,an o-nitrobenzyl N-cyclohexyl carbamate, an N-cyclohexyl-2-naphthalenesulfonamide, a 3,5-dimethoxybenzyl N-cyclohexyl carbamate, anN-cyclohexyl p-toluene sulfonamide; and a dibenzoin isophoronedicarbamate. In some embodiments, the photobase generator is selectedfrom the group consisting of: the photoactive group of PM3 and thephotoactive group of PM4.

In some embodiments, the photoactive group is cleaved upon exposure toan activating radiation. In some embodiments, the cleavage is homolyticcleavage. In some embodiments, the photoactive group initiates adownstream reaction upon exposure to an activating radiation.

In some embodiments, the photoactive group comprises an ionic organicsalt. In some embodiments, the photoactive group comprises an oniumsalt.

In some embodiments, the probe comprises a polypeptide. In someembodiments, the photoactive group is bound to said polypeptide. In someembodiments, the photoactive group is bound to a histidine side chain, aproline side chain, or a tyrosine side chain of said polypeptide.

In some embodiments, the probe is a polynucleotide or a singlenucleotide. In some embodiments, the photoactive group is bound to anucleobase of said polynucleotide or said single nucleotide. In someembodiments, the photoactive group is bound to a 2′ or 5′ carbon of saidpolynucleotide or said single nucleotide. In some embodiments, thesingle nucleotide comprises a removable blocking group. In someembodiments, the single nucleotide comprises a dideoxy terminator.

In some embodiments, the probe is selected from the group consisting of:a protein, a polypeptide, a glycoprotein, an oligosaccharide, and aglycolipid. In some embodiments, the probe is an antibody.

In some embodiments, provided herein is a composition comprising anucleotide according to Formula I:

wherein

n is from 0-3;

X is selected from the group consisting of: H, OPg, and a photoactivegroup, where Pg is a protecting group;

A is NH when

and A is N when

E is O when

and E is NHZ when

and

each Z is independently selected from the group consisting of: H, Me,and a photoactive group;

wherein at least one of said Z or X is said photoactive group.

In some embodiments, provided herein is a composition comprising anucleotide according to Formula II:

wherein

n is from 0-3;

X is selected from the group consisting of: H, OPg, and a photoactivegroup, where Pg is a protecting group;

A is NH when

and A is N when

E is O when

and E is NHZ when

and

each Z is independently selected from the group consisting of: H, Me,and a photoactive group;

wherein at least one of said Z or X is said photoactive group.

Also provided herein, according to some embodiments, is a compositioncomprising a modified nucleotide comprising a photoacid or photobasegenerator.

Also provided herein, according to some embodiments, is a compositioncomprising a modified nucleotide selected from the group consisting of:PM1, PM2, PM3, PM4, PM5, PM6, PM7, and PM8.

In some embodiments, the modified nucleotide is bound to a removableblocking group. In some embodiments, the removable blocking group is areversible terminator.

Also provided herein, according to some embodiments, is a polynucleotidecomprising the modified nucleotide comprising a photoactive groupprovided herein.

Also provided herein, according to some embodiments, is an arraycomprising a one or more polynucleotides comprising the modifiednucleotide comprising the photoactive group as provided herein, whereinthe one or more polynucleotides are immobilized to the surface of thearray. In some embodiments, the polynucleotide comprises PNA or LNA. Insome embodiments, the array comprises a reaction area comprising saidpolynucleotide, wherein said reaction area comprises a set of electrodesfor electronic monitoring of the pH of a solution. In some embodiments,the array comprises at least 100, at least 1,000, or at least 10,000 ofsaid reaction areas.

Also provided herein, according to some embodiments, is a substratecomprising one or more compositions comprising the modified nucleotidecomprising the photoactive group as provided herein, wherein thecomposition is immobilized to the surface of the substrate. In someembodiments, the substrate comprises an electrosensor capable ofdetecting a signal from said probe. In some embodiments, theelectrosensor is an ion-sensitive field effect transistor.

Also provided herein, according to some embodiments, is an array ofprobes comprising the modified nucleotide comprising the photoactivegroup as provided herein.

Also provided herein, according to some embodiments, is a system anarray of probes comprising the modified nucleotide comprising thephotoactive group as provided herein, wherein said array is inelectronic communication with a reader configured to receive anelectronic signal from said set of electrodes.

Also provided herein, according to some embodiments, is a method fordetecting a target biomolecule, comprising: providing probe capable ofbinding specifically to a target biomolecule, wherein said probe isbound to a photoacid generator or a photobase generator; contacting asample suspected of comprising said target biomolecule with said probe;removing unbound probes from said sample; exposing said sample to anwavelength of light capable of activating said photoacid generator orsaid photobase generator, such that said probe, if bound to said targetbiomolecule, releases an acid or a base upon exposure to said wavelengthof light; and detecting a concentration of ions in the sample, therebyidentifying the presence or absence of said target analyte based on achange of said concentration of ions.

In some embodiments, the probe comprises a polynucleotide or apolypeptide. In some embodiments, the probe is an antibody.

In some embodiments, the concentration of ions is determined bymeasuring an ionic strength of the sample. In some embodiments, theionic strength is measured using an ion-sensitive field effecttransistor.

In some embodiments, the sample is immobilized on the surface of asubstrate. In some embodiments, the substrate is an array. In someembodiments, the array comprises a plurality of wells, wherein saidwells each comprise a sensor for detecting an ionic strength of asolution in said wells. In some embodiments, the sensor is anion-sensitive field effect transistor.

Also provided herein, according to some embodiments, is a method ofdetecting a sequence identity of a target polynucleotide, comprising:providing a substrate comprising an immobilized target polynucleotidehybridized to a primer or probe; contacting said immobilized targetpolynucleotide with a solution comprising reagents for performing apolymerase extension reaction, said solution comprising a set ofmodified nucleotides comprising a photoactive group and a blockinggroup; exposing said substrate to conditions to promote incorporation ofone of said modified nucleotides at the 3′ end of said primer or probe;washing said substrate to remove unbound modified nucleotides; exposingsaid immobilized target polynucleotide to a wavelength of light toinduce said photoactive group to generate an acid or a base, therebygenerating a detectable change in ion concentration in a solutionsurrounding said immobilized target polynucleotide if said modifiednucleotide is incorporated into said target polynucleotide; detectingsaid change in ion concentration; and determining a sequence identity ofsaid target polynucleotide from said detected change in ionconcentration.

Also provided herein, according to some embodiments, is a method ofdetermining a sequence of a target polynucleotide, comprising: providingan array comprising a plurality of wells, wherein said wells comprise atarget polynucleotide to be sequenced bound to a surface of said well,and wherein said plurality of wells each comprise a sensor for detectingan electronic signal from said wells; performing a sequencing reactioncomprising performing at least one cycle, each cycle comprising:contacting said wells with a solution comprising reagents for performinga polymerase extension reaction, said solution comprising a set ofmodified nucleotides comprising a photoactive group and a removableblocking group; exposing said well to conditions to promoteincorporation of one of said modified nucleotides at the 3′ end of aprimer or probe hybridized to said single polynucleotide; washing saidwell to remove unbound modified nucleotides; exposing said well to awavelength of light to induce said photoactive group to generate andacid or a base, thereby generating a detectable change in ionconcentration; detecting the change in ion concentration with saidsensor; and if another cycle of the sequencing reaction is to beperformed, removing said terminator from said incorporated nucleotide.

In some embodiments, the electronic signal is specific to the identityof the base of the modified nucleotide added to the primer at eachcycle. In some embodiments, the electronic signal represents the pH of asolution in said well. In some embodiments, the electronic signal isanalyzed to determine a sequence of the target polynucleotide. In someembodiments, the sensor is an ion-sensitive field effect transistor.

In some embodiments, the photoactive group is photocleavable. In someembodiments, the photoactive group is a photoacid or photobasegenerator. In some embodiments, the removable blocking group is areversible terminator. In some embodiments, the reversible terminator isphotocleavable.

In some embodiments, the nucleotide set comprises only one of the groupconsisting of: nucleotides comprising adenine, nucleotides comprisingguanine, nucleotides comprising thymine, nucleotides comprisingcytosine, and nucleotides comprising uracil.

In some embodiments, the nucleotide set comprises nucleotides comprisingadenine, guanine, cytosine, and thymine or uracil. In some embodiments,the solution comprises a plurality of random primers. In someembodiments, the reagents for performing a polymerase extension reactioncomprise a primer capable of hybridizing to said single polynucleotide.

In some embodiments, if another cycle is to be performed, the methodfurther includes neutralizing the solution in the wells.

In some embodiments, the plurality of wells each comprise only a singletarget polynucleotide. In some embodiments, the plurality of wells eachcomprise a clonal population of a target polynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis insteadplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 shows a general structure of a modified nucleotide comprising aphotoactive group at the 2′ carbon, according to some embodiments.

FIG. 2 shows embodiments of covalently bound attachment points ofphotoactive groups to different nucleobases to make a modifiednucleotide.

FIG. 3 shows examples of photoactive groups bound to a nucleobase withor without a linker.

FIG. 4 shows examples of salts including photoactive groups bound to anucleobase.

FIG. 5 shows examples of photoactive groups bound at the 2′ or 5′position of the ribose component of a nucleotide.

FIGS. 6A and 6B shows embodiments of a removable blocking group (e.g., areversible terminator) bound to a 3′ carbon of a sugar moiety on anucleotide (FIG. 6A) or to the base of a nucleotide (FIG. 6B).

FIG. 7 shows steps in a sequencing by synthesis reaction using themodified nucleotides comprising photobase or photoacid generators,according to an embodiment of the invention (SEQ ID NO: 27).

FIG. 8 shows results of a DNA sequencing reaction using modifiednucleotides described herein and detected by monitoring pH. Thesequential cycled addition of nucleotides to a growing strand generatesa sequence of signals which corresponds to the sequence of thesynthesized oligonucleotide or the template strand (SEQ ID NO: 27).

FIG. 9 provides an example of such an array comprising a plurality ofISFET sensors arranged along a grid.

FIG. 10 depicts an exemplary single nucleotide primer extension reactionto detect a sequence variant (SEQ ID NOS 28-29, respectively, in orderof appearance).

FIG. 11 shows a diagram of a system including a device that interfaceswith a chip or array as described herein to collect data from the chipand process it. Examples of mechanisms to obtain information from eachof the multiple reaction areas on the array are shown.

FIG. 12 shows a plate base contact to plate base readout stage andelements for UV exposure to induce photoactivation or photocleavage,according to an embodiment of the invention.

FIG. 13 is a network diagram of an example system environment includingthe sequencing system in communication with one or more client devicesand one or more servers via a network.

FIG. 14 shows a pathway for synthesis of dCTP-PAG1 (PM1).

FIG. 15 shows a pathway for synthesis of dATP-PAG2 (PM2).

FIG. 16 shows a pathway for synthesis of dUTP-PBG1 (PM3).

FIG. 17 shows a pathway for synthesis of dGTP-PBG2 (PM4).

FIG. 18 shows a table of results of an assay to detect incorporation ofeach of the four modified nucleotides into a sequence.

FIGS. 19A, 19B, 19C and 19D show a graph of results of an assay todetect incorporation of each of the four modified nucleotides into asequence.

FIG. 20 shows the structure of modified nucleotide ddCTP-PAG1 (PM5).

FIG. 21 shows the structure of modified nucleotide ddATP-PAG2 (PM6).

FIG. 22 shows the structure of modified nucleotide ddUTP-PBG1 (PM7).

FIG. 23 shows the structure of modified nucleotide ddGTP-PBG2 (PM8).

FIGS. 24A and 24B show the results of detection of incorporation andidentity of a single modified nucleotide (for each of PM5-PM8) forsequencing.

DETAILED DESCRIPTION

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein, the term “photoactive group” refers to a functionalgroup that undergoes a lysis reaction when exposed to electromagneticradiation, heat, or an initiator species, thereby generating an acid ora base. Compounds or functional groups that generate an acid whenexposed to electromagnetic radiation within a spectrum of wavelengthand/or energy are referred to herein as photoacid generators. Compoundsor functional groups that generate an acid when exposed toelectromagnetic radiation within a spectrum of wavelength and/or energyare referred to herein as photobase generators. As used herein,photoactive groups can be bound to probes, such as antibodies,polynucleotides, or incoming pairing nucleotides duringsequence-directed polymerization such that the compounds can bespecifically detected due to the generation of an acid or a base whenexposed to an activating radiation.

As used herein, the terms “photoactive compound” or “photoactivemolecule” refer to an organic compound or molecule comprising aphotoactive group. In some embodiments, an organic compound comprising aphotoactive functional group undergoes a lysis reaction upon exposure toradiation to generate an acid or a base. In some embodiments, the lysisreaction is a homolysis reaction. In some embodiments, a photoactivegroup or compound converts electromagnetic radiation into chemicalenergy and may need an initiator to start the photochemical or otherwisehomolysis reaction, i.e. a compound is added to the composition thatinitiates homolysis by reacting with the electromagnetic radiation, oreven heat, to form an intermediate initiating species, e.g., freeradicals or cations, that react further with the photoactive group. Theradical product from an initiator need not react directly, or next insequence with a photoactive group. The initiating species may react withanother compound in a chain reaction to produce the desired chemicalreaction with a photoactive group.

Photoactive compounds or groups include, for example, cationicphotoinitiators such as photoacid generators (PAGs) or photobasegenerators (PBGs), which generate a corresponding photoacid orphotobase, respectively, when exposed to electromagnetic radiation.Examples of photoactive compounds are disclosed in the InternationalPatent Publication No. WO/2014/078606, “Substrates, Systems, and Methodsfor Array Synthesis and Biomolecular Analysis,” filed Nov. 14, 2013,which is incorporated herein in its entirety for all purposes. Aphotoinitiator is a compound especially added to a formulation toconvert electromagnetic radiation into chemical energy in the form ofinitiating species, e.g., free radicals or cations. The acid, base, orother product of a photoactive compound exposed to electromagneticradiation may then react with another compound in a chain reaction toproduce a desired chemical reaction which can then be detected.

As used herein the terms “polypeptide,” “peptide,” or “protein” are usedinterchangeably to describe a chain or polymer of amino acids that arelinked together by bonds. Accordingly, the term “peptide” as used hereinincludes a dipeptide, tripeptide, oligopeptide, and polypeptide. Theterm “peptide” is not limited to any particular number of amino acids.In some aspects, a peptide contains about 2 to about 50 amino acids,about 5 to about 40 amino acids, or about 5 to about 20 amino acids. Amolecule, such as a protein or polypeptide, including an enzyme, can bea “native” or “wild-type” molecule, meaning that it occurs naturally innature; or it may be a “mutant,” “variant,” “derivative,” or“modification,” meaning that it has been made, altered, derived, or isin some way different or changed from a native molecule or from anothermolecule such as a mutant.

As used herein the term “biomolecule” refers to any molecule(s) thatoccur naturally in a living organism. As such, the term biomoleculesincludes macromolecules such as, but certainly not limited to: proteins,carbohydrates, lipids and nucleic acids; and further also smallmolecules such as precursors and metabolites including, but not limitedto: L-lysine, selenocysteine, isoprene, ATP and tocopherol.

As used herein, the term “probe molecules” refers to, but is not limitedto, peptide nucleic acids (“PNA”), DNA binding sequences,oligonucleotides, nucleic acids, deoxyribonucleic acids (DNA),ribonucleic acids (RNA), nucleotide mimetics, chelates, side-chainmodified peptide sequences, biomarkers and the like. As used herein, theterm “feature” refers to a particular probe molecule that has beenattached to a microarray. As used herein, the term “ligand” refers to amolecule, agent, analyte or compound of interest that can bind to one ormore features.

As used herein the term “linker molecule” or “spacer molecule” includesany molecule that does not add any functionality to the resultingbiomolecule but spaces and extends out the biomolecule from thesubstrate, thus increasing the distance between the substrate surfaceand the growing peptide, nucleic, or in general the growing biomolecule.This generally reduces steric hindrance with the substrate for reactionsinvolving the biomolecule (including uni-molecular folding reactions andmulti-molecular binding reactions) and so improves performance of assaysmeasuring one or more aspects of functionality.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is a specific antibody/immunoglobulin molecule.

As used herein the term “antibody” or “immunoglobulin molecule” refersto a molecule naturally secreted by a particular type of cells of theimmune system: B cells. There are five different, naturally occurringisotypes of antibodies, namely: IgA, IgM, IgG, IgD, and IgE.

The term “antigen” as used herein refers to a molecule that triggers animmune response by the immune system of a subject, e.g., the productionof an antibody by the immune system. Antigens can be exogenous,endogenous or auto antigens. Exogenous antigens are those that haveentered the body from outside through inhalation, ingestion orinjection. Endogenous antigens are those that have been generated withinpreviously-normal cells as a result of normal cell metabolism, orbecause of viral or intracellular bacterial infection. Auto antigens arethose that are normal protein or protein complex present in the hostbody but can stimulate an immune response.

As used herein the term “epitope” or “immunoactive regions” refers todistinct molecular surface features of an antigen capable of being boundby component of the adaptive immune system, e.g., an antibody or T cellreceptor. Antigenic molecules can present several surface features thatcan act as points of interaction for specific antibodies. Any suchdistinct molecular feature can constitute an epitope. Therefore,antigens have the potential to be bound by several distinct antibodies,each of which is specific to a particular epitope. biological sample

As used herein, the term “wafer” refers to a slice of semiconductormaterial, such as a silicon or a germanium crystal generally used in thefabrication of integrated circuits. Wafers can be in a variety of sizesfrom, e.g., 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimensionwith thickness from, e.g., 275 μm to 775 μm.

As used herein the term “microarray,” “array,” or “chip” refers to asubstrate on which different probe molecules of protein or specific DNAbinding sequences have been affixed at separate locations in an orderedmanner thus forming a microscopic array. In some embodiments, specificPNA, RNA or DNA binding sequences have been affixed at separatelocations in an ordered manner thus forming a microscopic array.Specific PNA, RNA or DNA binding sequences may be bound to the substrateof the chip through one or more different types of linker molecules. A“chip array” refers to a plate having a plurality of chips, for example,24, 96, or 384 chips.

As used herein the term “microarray system” refers to a system usuallycomprised of bio molecular probes formatted on a solid planar surfacelike glass, plastic or silicon chip plus the instruments needed tohandle samples (automated robotics), to read the reporter molecules(scanners) and analyze the data (bioinformatic tools).

As used herein the terms “substrate” and “solid support” are usedinterchangeably and refer to any insoluble, polymeric material. Suchmaterials must have rigid or semi-rigid surface, where examples include,but are not limited to, natural polymeric materials such as glass orcollagen, and synthetic polymers such as acrylamide, polyvinyl chordae,or silicon based arrays.

As used herein, the term “PNA-DNA chimera” refers to an oligomer, oroligomers, comprised of: (i) a contiguous moiety of PNA monomer unitsand (ii) a contiguous moiety of nucleotide monomer units with anenzymatically-extendable terminus

As used herein, the term “primer extension” refers to an enzymaticaddition, i.e., polymerization, of monomeric nucleotide units to aprimer while the primer is hybridized (annealed) to a template nucleicacid.

As used herein, the term “assay” refers to a type of biochemical testthat measures the presence or concentration of a substance of interestin solutions that can contain a complex mixture of substances.

As used herein, the term “activating radiation” refers toelectromagnetic radiation of a defined wavelength and energy sufficientto activate a photoactive group or compound to induce a reaction. Inpreferred embodiments, this reaction is the release of an acid or a basefrom a photoacid generator or a photobase generator, respectively.

As used herein, the term “blocking group” refers to a moiety bound to amonomer that prevents incorporation of a subsequent monomer in thesynthesis of a polymer. A removable blocking group is one that can beremoved to provide a binding site for incorporation of the next monomer.Removable blocking groups are commonly used for sequencing-by-synthesisreactions to control the stepwise addition of nucleotides during atemplate-directed polymerization reaction.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Overview

Described herein, according to some embodiments, are methods andcompositions for sensitive and specific detection of target biomoleculesimmobilized to the surface of a substrate using probes tagged withphotoactive compounds. As described herein, in some embodiments, thephotoactive compounds are photoacid or photobase generators thatgenerate a measurable change in the pH of the surrounding solution uponexposure to a wavelength of light. This change in pH is specific to thetype of photobase or photoacid generator, such that multiple photoactivecompounds can be distinguished. These probes comprising photoactivegroups facilitate detection methods that provide a highly sensitive andspecific detection that is not limited by the constraints of detectionfrom light. In some embodiments, these methods are performed on an arrayof ISFET detectors to measure the change in pH of the solutionsurrounding bound probes comprising a photoactive group. Synthesis ofseveral embodiments of probes bound to photoactive groups and the use ofthe same for detection of target biomolecules, including for sequencediscrimination and sequence determination, is provided herein.

Photoactive Compounds

Disclosed herein are photoactive groups, i.e. photoactive organicmolecules or functional groups. In a most general sense, photoactivegroups or compounds are those which possess at least one chemical moietythat becomes reactive when exposed to radiation such as ultraviolet orvisible light. Exposure of the photoactive compounds to electromagneticradiation is a primary photochemical event that produces a change in thepH of the surrounding microenvironment. This change is brought about bythe acidic or basic chemical species that is produced due tophotoactivation of the photoactive group or compound. One or morephotoactive group or compound may react by an elimination, addition, orrearrangement reaction; and may require an optional additive, orinitiator, to kick-off the reaction. In some embodiments,photoactivation generates a homolysis reaction to generate an acid or abase. In some embodiments, the photoactive groups or photoactivecompounds are photoacid generators or photobase generators that directlygenerate an acid or a base upon photoactivation, e.g., from a homolysisreaction. In some embodiments, the photoactive groups or photoactivecompounds are photoinitiators that indirectly release an acid or a base,e.g., through release of a chemical species that reacts downstream withanother species to release an acid or a base.

Generally, the skilled artisan can easily identify a given functionalgroup as a photoacid generator or a photobase generator since only thosegroups will form an organic acid or base possessing a proton orheteroatom that is recognizable as an acidic or basic group uponhomolysis of the bond attaching that group to the compound. In thisregard, the skilled artisan quickly recognizes a photoacid generator ora photobase generator by working backwards (in a sense) in identifyingan acidic or basic functional group. For example, tertiary aminefunctional groups are recognized by organic chemists to be significantlybasic because there is more electron density on the nitrogen atom of atertiary amine, as opposed to say a secondary amine. Accordingly, askilled artisan would recognize that any compound that has a quaternaryamine functional group that will, upon homolysis, form a tertiary aminefunctional group, is a photoactive compound or functional group of thepresent disclosure.

In some embodiments, photoactive compounds or functional groups of thepresent disclosure will only include those which produce a compoundhaving a pKa that is significantly acidic or basic so that one skilledin the art would recognize that a veritable organic acid or base wouldbe generated. To this end, in some embodiments, photoactive compounds orfunctional groups of the present disclosure produce an organic compoundthat has a pKa of 10 or higher, 11 or higher, or 12 or higher.Photoactive compounds or functional groups of the present disclosureinclude acids or bases that are recognized in the art as “hard” or“soft”.

In some aspects, photoactive compounds or functional groups of thepresent disclosure also have acidity or basicity according to the energyof their lowest unoccupied molecular orbital (LUMO) and/or energy oftheir highest unoccupied molecular orbital (HOMO). In some embodiments,the photoactive compounds or functional group produces a photoacid thathas LUMO energy of −2.5 eV or lower (this is in terms of energy, so +1would be lower). In some embodiments, the photoactive compounds orfunctional group produces a photobase that has HOMO energy of 1.7 eV orhigher (this is in terms of energy, so −3 would be higher).

Photoactive compounds comprise at least one photoactive group to convertabsorbed light energy, UV or visible light, into chemical energy in theform of initiating species, e.g., free radicals or cations. As such, ina general aspect, the photoactive groups or compounds of the presentdisclosure can be any organic functional group or compound thatpossesses one or groups that will absorb energy anywhere from 200 nm to700 nm.

In general, photoactive compounds or functional groups are known to oneskilled in the art. Examples of photoactive compounds or functionalgroups that are photoacid generators (PAG) include, but in no way arelimited to: sulfonium salts, iodonium salts, sulfonyldiazomethane,N-sulfonyloxyimide, benzoinsulfonate, nitrobenzylsulfonate, sulfone,glyoxime derivatives, halogenated triazines, onium salts such asaryldiazonium salts and diaryl halonium salts, triaryl sulfonic salts,sulfonated esters, substituted hydroxyimides, substitutedhydroxylimines, azides, naphthoquinones such as diazonaphthoquinones,diazo compounds, and many combinations thereof, nitrobenzyl esters,sulfones, phosphates, and the like. Examples of photoactive compounds orfunctional groups that are photobase generators (PBG) include, but in noway are limited to: o-acyloximes, benzoyloxycarbonyl derivatives,photoactive carbamates such as benzyl carbamates and benzoin carbamates,oxime ester compounds like o-carbamoyloximes, ammonium compounds likequatemary ammonium tetraphenyl borate salts, benzoin compounds,dimethoxybenzyl urethane compounds, orthonitrobenzyl urethane compounds,aromatic sulfonamides, alpha-lactams, N-(2-arylethenyl) amides, mixturesthereof, and the like. These compounds generally generate amine basesafter irradiation. Photobase generators can also generate ammonia orhydroxy ions due to the action of light may also be used. These can beselected from, for example, N-substituted4-(o-nitrophenyl)dihydroxypyridines,N-(2-nitrobenzyloxycarbonyl)piperidine,1,3-bis(N-(2-nitrobenzyloxycarbonyl)-4-piperidyl]propane,N,N′-bis(2-nitrobenzyloxycarbonyl)dihexylamine, andO-benzylcarbonyl-N-(1-phenylethylidene)hydroxylamine. A good review ofphotoacid and photobase generators is found, for example, in Prog.Polym. Sci. vol. 21, 1-45, 1996, the entire contents and disclosure ofwhich is incorporated herein by reference. Very specific examples of asuitable photobase generators include, but are not limited to:2-hydroxy-2-phenylacetophenone N-cyclohexyl carbamate (i.e.,C₆H₅C(═O)CH(C₆H₅)OC(═O)NHC₆H₁₁); o-nitrobenzyl N-cyclohexyl carbamate(i.e., o-NO₂C₆H₅CH₂C(═O)NHC₆H₁₁); N-cyclohexyl-2-naphthalene sulfonamide(i.e., C₁₀H₇SO₂NHC₆H₁₁); 3,5-dimethoxybenzyl N-cyclohexyl carbamate(i.e., (CH₃O)₂C₆H₅CH₂C(═O)NHC₆H₁₁); N-cyclohexyl p-toluene sulfonamide(i.e., p-CH₃C₆H₅SO₂NHC₆H₁₁); and dibenzoin isophorone dicarbamate.Finally, photoactive compound or functional group also includes anycompounds or functional groups that behave as both photobases andphotoacids. These compounds are described in the art as single componentphotoacid/photobase generators.

In some embodiments, a photoactive compound or group can be a photoacidgenerator (PAG) or a photobase generator (PBG). Photoacid generators (orPAGs) are cationic photoinitiators. A photoinitiator is a compoundespecially added to a formulation to convert absorbed light energy, UVor visible light, into chemical energy in the form of initiatingspecies, e.g., free radicals or cations. Cationic photoinitiators areused extensively in optical lithography. The ability of some types ofcationic photo initiators to serve as latent photochemical sources ofvery strong protonic or Lewis acids is generally the basis for their usein photo imaging applications.

In some embodiments, a photoacid generator is an iodonium salt, apolonium salt, or a sulfonium salt. In some embodiments, a photoacidgenerator is (4-Methoxyphenyl)phenyliodonium ortrifluoromethanesulfonate. In some embodiments, a photoacid generator is(2,4-dihydroxyphenyl)dimethylsulfonium triflate or (4methoxyphenyl)dimethylsulfonium triflate, shown below:

In some embodiments, a photoacid generator is iodonium and sulfoniumsalts of triflates, phosphates and/or antimonates.

In some embodiments, a photobase generator is1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane or1,3-Bis[(1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane.

Conjugation of Photoactive Groups to Compounds

Generally the methods used to conjugate one or more photoactivemolecule(s) or functional group(s) are known in the art. The skilledartisan will appreciate that various types of carbon-carbon andcarbon-heteroatom bonds can be made that will attach a given photoactivemolecule or functional group to any such biomolecule of interest. Ofcourse the skilled artisan will appreciate that any such attachment mustnot interfere with the binding, enzymatic and/or biological function ofone or more biomolecule(s) described herein, i.e. the activity of thebiomolecule it is attached to must not be rendered inoperative nor shallthe activity be rendered inoperative of other biomolecules such as DNApolymerase and the like that are otherwise present. To this end, oneskilled in the art can refer to texts.

Further, once the photoactive compounds are synthesized, the task ofconjugating these compounds onto either the nitrogenous base, saccharideresidue, or phosphate is within the purview of one skilled in the art oforganic synthesis. There is much literature and even whole texts thatare now that review and are able to instruct an organic chemist how toperform the synthetic methods needed for “conjugating” a small organicmolecule to an activated position on a biomolecule or even incorporatinga “conjugated” or “tagged” (fluorescent or otherwise) molecule into abiological process, such as enzymatic bond cleavage or construction.Such synthetic methods nowadays are routine to the trained organic ormedicinal chemist. Relevant review articles, texts, and books include,but are not limited to: Hermanson, G. T., Bioconjugate Techniques,3^(rd) Ed., Academic Press, Oxford (2013); Sinh, Y. et al., “Recentdevelopments in oligonucleotide conjugation” Chem. Soc. Rev., 2010, 39,2054-2070; and Roy, B. et al., “Recent Trends in Nucleotide Synthesis”Chem. Rev., 2016, 116 (14), pp 7854-7897.

As a general guide, herein disclosed are representative syntheses toattach a photoactive molecule or functional group to a givenbiomolecule. As such, these synthetic techniques may not cover each andevery way to conjugate a photoactive molecule and thus, are not meant tobe limiting in any form since the skilled artisan will be able to usealternative synthetic reactions to conjugate or otherwise insert aphotoactive molecule or functional group to a given biomolecule onto abiomolecule.

Photoactive Nucleotides

In some embodiments, the photoactive group is covalently ornon-covalently attached to one or more nucleotides, or any combinationthereof. In some embodiments, the photoactive group is covalently in aselective manner. For example, the photoactive group may be covalentlyattached to a guanine nucleobase/nucleotide, but not any othernucleobases/nucleotides. In another example, the photoactive group maybe covalently attached to a thymine nucleobase/nucleotide, but not anyother nucleobases/nucleotides. In another example, the photoactive groupmay be covalently attached to any combination of two (2) of: cytosine,guanine, adenine, thymine and uracil (C, G, A, T, and U, respectively)nucleobases/nucleotides, but not any other nucleobases/nucleotides. Inanother example, the photoactive group may be covalently attached to anycombination of three (3) of: cytosine, guanine, adenine, thymine anduracil (C, G, A, T, and U, respectively) nucleobases/nucleotides, butnot any other nucleobases/nucleotides. In another example, thephotoactive group may be covalently attached to any combination of four(4) of: cytosine, guanine, adenine, thymine and uracil (C, G, A, T, andU, respectively) nucleobases/nucleotides, but not any othernucleobases/nucleotides.

In one embodiment, nucleotides of the present disclosure are tagged witha photoactive group. In one embodiment nucleotides of the presentdisclosure have a general structure according to FIG. 1. The photoactivegroup can be bound to the sugar, i.e., at the 2′ C position. Thephotoactive group can also be bound to the nucleobase in a way that doesnot interfere with hydrogen bonding to its cognate nucleobase.

In a general embodiment, the photoactive group is covalently attached toa nucleobase to make a modified nucleotide. In such an embodiment, thephotoactive group may even be attached at a nitrogen atom of anucleobase. Though it is preferable that the modification retain thebiologically activity of the nucleotide, for example an enzyme such DNApolymerase I would still be capable of using the tagged nucleotide tocreate one or more complimentary strands of RNA or DNA or the like. Insuch embodiments, the tag, i.e. the photoactive group, may generally becovalently attached to the nucleobase according to the FIG. 2.

In some embodiments, the photoactive group is covalently attached to anucleobase via a linker. The linker can be from 2-40 atoms in length andneed not be restricted in terms of the identity of heteroatoms andcarbon atoms that it comprises. More to this point, the identity of theparticular organic functional groups that comprise the linker is notcrucial or limiting as long as one or more of the functional groupsthemselves are photoactive groups and do not react with other functionalgroups of the nucleotide(s) or other components in the system so as torender them inoperable, e.g. peptide side chain functional groups arenot altered so as to impact a loss in biological activity or even anenzyme, present in the system/assay, perhaps DNA polymerase I is notaffected so as its function is impaired. Additionally in someembodiments, the photoactive group is covalently attached to anucleobase directly and without any functional group as a linker.Examples of these photoactive group tags covalently bonded to anucleobase are shown in FIG. 3 and are not meant to be limiting in anyway.

In other such embodiments, the tag may even be attached in such a way asto create an onium salt or any other otherwise ionic organic salts thatcan release acidic or basic species upon homolysis. Examples of suchsalts include photoactive groups covalently bonded in FIG. 4.

In other embodiments, the photoactive group is covalently attached tothe five carbon saccharide at the 2-position. The photoactive group neednot be covalently bonded thru an oxygen atom at the 2-position, rather acarbon-carbon, carbon-nitrogen, or even a carbon-sulfur bond may beconstructed and the photoactive group tag may be covalently bonded onthe substituent at that is connected at the 2-position. Somenon-limiting examples of tags at the 2-position include those in FIG. 5.

In other embodiments, the photoactive group need not be covalentlyattached thru the nucleobase of a nucleotide and instead, can becovalently attached thru the 3′ position, covalently bonded to thehydroxyl or not, otherwise alternatively covalently bonded thru thecarbon atom at the 3′ position, on the saccharide residue or covalentlyattached to an oxygen atom on the phosphate group of a nucleotide. Inother embodiments, the photoactive group is non-covalently bonded to anoxygen atom on the phosphate group of a nucleotide.

Alternatively, the photoactive group can be attached non-covalently thrua chelate to one or more non-terminal nucleotides. For instance if thenucleotide is modified to have a linker functional group, say ethylenediamine, that forms an cooper (Cu) metal atom chelate, a photoactivegroup may be non-covalently attached to the nucleotide via the metalchelate.

Nucleotides can be modified to attach a photoactive molecule orfunctional group on either a carbon atom or a nitrogen atom of thenitrogenous base (so long as the covalent bond to the nitrogen atom doesnot adversely affect the biological activity, structural integrity, orrecognition of the nucleotide). A couple of examples reactions bonding aphotoactive molecule to the nitrogenous base of a nucleotide follow.

In one embodiment, a nucleotide is covalently bonded thru a linker tothe photoactive group. In such an embodiment, the linker can be from one(1) to forty (40) atoms in length.

In some embodiments, the modified nucleotides described herein areincorporated into a polynucleotide probe that can bind specifically viahybridization to a target biomolecule comprising a polynucleotidesequence complementary to a portion of said polynucleotide probe. Thephotoactive group can be placed at a specific region of thepolynucleotide probe. In some embodiments, the photoactive group ispositioned at the end of the polynucleotide probe. In some embodiments,a polynucleotide probe comprises multiple photoactive groups placed atmultiple positions along the nucleotide sequence.

Removable Blocking Groups

In some embodiments, the modified nucleotides comprise blocking groupsthat prevent addition of more than one modified nucleotide during areaction to polymerize a growing strand of an oligonucleotide. In someembodiments, the blocking group is a fixed terminator group, such as adideoxy terminator. In some embodiments, the blocking group is aremovable blocking group, such as a reversible terminator. In someembodiment, the removable blocking group is photocleavable such that,upon exposure to light, it is removed to allow subsequent addition ofnucleotides to the growing strand. In some embodiments, the modifiednucleotide comprises a 3′-bound removable blocking group (FIG. 6A) wherethe blocking group —OR is linked to the oxygen atom of the 3′-OH of thepentose, while the photoactive group is linked to the base and is usedfor detection of the base attached. In some embodiments, the removableblocking group is the same group or bound to the same region of thenucleotide as the photoactive group.

In general, removable blocking groups can be used insequencing-by-synthesis approaches that infer the sequence of a templateby stepwise primer elongation. The sequencing process can involve (i)immobilizing the template and primers on the wafer; (ii) primerextension by one base and termination; (iii) obtaining the pH reading toidentify the added nucleotide; (iv) removal of the blocking group thatprevents the following polymerase addition; (v) washing and repeatingthe steps (ii-iv).

Peptides Comprising Photoactive Groups

Peptides can be modified to attach a photoactive molecule or functionalgroup on the side chain. For example, the amine functional group on theside chain of a lysine amino acid residue in a given peptide sequencecan be used in a nucleophilic displacement reaction with anelectrophilic photoactive molecule. Such electrophilic species areeasily prepared, for examples, aryl alkyl esters can be modified to thecorresponding halo-aryl alkyl ester through an electrophilic aromaticsubstitution reaction. The halo-aryl alkyl ester is now an electrophileand can be used in an aromatic nucleophilic substitution reaction withthe amine functional group of the lysine residue to create a covalentbond between the nitrogen atom on the side chain of lysine to the carbonatom (where the halogen resided) on the aromatic ring of the photoactivemolecule.

Of course, covalent bonds are not the only means that are describedherein to bond one or more photoactive molecule(s) or functionalgroup(s). Ionic bonds, Van der Waals bonding and the like are alsoencompassed within the present disclosure. The skilled artisan willrecognize the functional groups on a given biomolecule that can providesuch bonding, e.g. a charged phosphonate group on, say, a nucleic acidmay be used for an ionic bond or a terminal carboxylate on a peptide maybe also be used (under basic conditions of course). The skilled artisanneed only consult a text or literature reference to use a syntheticmethod to make these bonds.

As such, a given covalent bond on the biomolecule may be made to anycarbon atom or heteroatom so long as that attachment does not alter thebiological function of the biomolecule. For example, a covalent bond mayattach a photoactive molecule or functional group to a nucleic acid.This bond may be attached thru say, a carbon atom on a guaninenitrogenous base to, for instance, an oxygen atom of the photoactivemolecule or functional group. This type of conjugation is well withinthe parameters of the present disclosure as long as the nucleic aciddoes not lose biological activity, or perhaps the nucleic acid sequencereadability with DNA polymerase.

An exemplary reaction scheme for binding a photoactive compound to aprotein is shown below:

Synthesis of Photoactive Groups or Compounds

Photoactive compounds or functional groups (i.e. organic functionalgroups), are needed as small molecules in the present disclosure and canbe purchased “off” the shelf from a petrochemical vendor such as SigmaAldrich, VWR, Fisher Scientific, etcetera or can be easily synthesizedby the skilled artisan trained in classical organic syntheses. Thebreadth of small organic molecules (or functional groups) that areencompassed within the present disclosure is relatively large. However,one skilled in organic synthesis will instantly identify numerousclassical reactions that will produce the requisite molecule fromreadily available starting material. Apropos, many of the photoactivemolecules (or functional groups), and the covalent bonds needed tosynthesize them, are single step preparations or otherwise relativelyfacile chemistry for those in the art.

The compounds described herein can be prepared by any of the applicabletechniques of organic synthesis. Many such techniques are well known inthe art. However, many of the known techniques are elaborated inCompendium of Organic Synthetic Methods (John Wiley & Sons, New York)Vol. 1, Ian T. Harrison and Shuyen Harrison (1971); Vol. 2, Ian T.Harrison and Shuyen Harrison (1974); Vol. 3, Louis S. Hegedus and LeroyWade (1977); Vol. 4, Leroy G. Wade Jr., (1980); Vol. 5, Leroy G. WadeJr. (1984); and Vol. 6, Michael B. Smith; as well as March, J., AdvancedOrganic Chemistry, 3rd Edition, John Wiley & Sons, New York (1985);Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency inModern Organic Chemistry, In 9 Volumes, Barry M. Trost, Editor-in-Chief,Pergamon Press, New York (1993); Advanced Organic Chemistry, Part B:Reactions and Synthesis, 4th Ed.; Carey and Sundberg; KluwerAcademic/Plenum Publishers: New York (2001); Advanced Organic Chemistry,Reactions, Mechanisms, and Structure, 2nd Edition, March, McGraw Hill(1977); Protecting Groups in Organic Synthesis, 2nd Edition, Greene, T.W., and Wutz, P. G. M., John Wiley & Sons, New York (1991); andComprehensive Organic Transformations, 2nd Edition, Larock, R. C., JohnWiley & Sons, New York (1999).

The skilled artisan can easily identify a given functional group asphotoactive since only those groups will form an organic acid or basepossessing a proton or heteroatom that is recognizable as an acidic orbasic group upon homolysis of the bond attaching that group to thecompound. In this regard, the skilled artisan quickly recognizes aphotoactive group by working backward to identify functional groups in asense. For example, tertiary amine compounds are recognized by organicchemists to be an organic base because there is more electron density onthe nitrogen atom of a tertiary amine, as opposed to a secondary amine.Apropos, a skilled artisan would recognize that a compound that has aquaternary amine functional group that will, upon homolysis, form atertiary amine functional group, is a photoactive compound or functionalgroup of the present disclosure. Of course, most any functional groupcan be considered, to some extent, acidic or basic. Therefore,photoactive compounds or functional groups of the present disclosurewill only include those which have a significant pKa so that one skilledin the art would instantly recognize that an organic acid or base wouldbe generated. Thus, it is fair to generalize that the organic functionalgroups acidic or basic for photochemical use herein generally, thoughnot always, include a bond to heteroatom. More specifically, theheteroatoms that include oxygen, nitrogen, sulfur, and the halogens areespecially attractive because homolysis of a compound containing theseheteroatoms will produce a compound that is acidic having anoxygen-hydrogen or halogen-hydrogen bond; or will be basic having a lonepair of electrons on a sulfur, oxygen, or nitrogen atom. Of course,these are not the extent of or entire list of heteroatoms that theskilled artisan will recognize as being potentially acidic or basic, butthese are, generally, speaking the most common. Such organic functionalgroups produced from the homolysis of a moiety having a covalent bond toa heteroatom include, but are not limited to: carboxylate esters (O—Cbond homolysis forming the requisite carboxylic acid), α-halogenatedethers (O—C bond homolysis forming the requisite α-haloalcohol), α-nitroethers (O—C bond homolysis forming the requisite α-nitroalcohol), phenylethers (O—C bond homolysis forming the requisite phenol), sulfonateesters (O—C bond homolysis forming the requisite organic sulfonic acid),phosphonate esters (O—C bond homolysis forming the requisite organicphosphonic acid), and anhydride (O—C bond homolysis forming therequisite organic carboxylic acid).

Of course, the skilled artisan will consult the relevant literature tosynthesize the requisite covalent bonds to produce photoactive compoundsor functional groups. Particularly attractive review articles that areon point include: Shirai, M.; Tsunooka, M., “Photoacid and photobasegenerators: chemistry and applications to polymeric materials” Progressin Polymer Science 1996, 21(1), 1-45.; Shirai, M.; Suyama, K.; Okamura,H.; Tsunooka, M., “Development of novel photosensitive polymer systemsusing photoacid and photobase generators” Journal of PhotopolymerScience and Technology 2002, 15(5), 715-730; Houlihan, F. M.; Neenan, T.X.; Reichmanis, E.; Kometani, J. M.; Chin, T., “Design, synthesis,characterization, and use of all-organic, nonionic photogenerators ofacid” Chemistry of Materials 1991, 3(3), 462-71; Ahmad Hasan,Klaus-Peter Stengele, Heiner Giegrichl, Paul Comwell, Kenneth R. Isham,Richard A. Sachleben, Wolfgang Pfleiderer, and Robert S. Foote,Photolabile Protecting Groups for Nucleosides: Synthesis andPhotodeprotection Rates, Tetrahedron, Vol. 53, No. 12, pp. 4247-4264,1997; Serafinowski and Garland, J Am. Chem. Soc. 2003, 125, 962-965;Iwashima, C.; Imai, G.; Okamura, H.; Tsunooka, M.; Shirai, M.,“Synthesis of i- and g-line sensitive photoacid generators and theirapplication to photopolymer systems” Journal of Photopolymer Science andTechnology 2003, 16(1), 91-96; Okamura, Haruyuki; Sakai, Koichi;Tsunooka, Masahiro; Shirai, Masamitsu; Fujiki, Tsuyoshi; Kawasaki,Shinich; Yamada, Mitsuaki. I-line sensitive photoacid generators andtheir use for photocrosslinking of polysilane/diepoxyfluorene blend.Journal of Photopolymer Science and Technology (2003), 16(1), 87-90;Okamura, Haruyuki; Sakai, Koichi; Tsunooka, Masahiro; Shirai, Masamitsu.Evaluation of quantum yields for decomposition of I-line sensitivephotoacid generators. Journal of Photopolymer Science and Technology(2003), 16(5), 701-706; and Okamura, Haruyuki; Matsumori, Ryosuke;Shirai, Masamitsu. I-line sensitive photoacid generators havingthianthrene skeleton. Journal of Photopolymer Science and Technology(2004), 17(1), 131-134.

The skilled artisan can also consult literature and/or textbooks onorganic syntheses involving carbon-heteroatom bond construction. Suchtexts include, but are not limited to: Yudin, A., CatalyzedCarbon-Heteroatom Bond Formation, Wiley-VCH Verlag, Weinheim, Germany(2011); Taber, D. and Lambert, T., Organic Synthesis: State of the Art2011-2013, Oxford Press, Oxford, (2015); and Wolfe, J., Synthesis ofHeterocycles via Metal-Catalyzed Reactions that Generate One or MoreCarbon-Heteroatom Bonds Springer, New York, (2013).

Forthwith disclosed are some very general synthetic methods, known inthe art, for constructing photoactive groups that include organicfunctional groups such as: alcohol, carboxylate esters, sulfonateesters, phosphonate esters, and ethers. Examples of these reactions orperhaps representative reaction schemes follow.

In one embodiment, one or more photoactive compounds or functionalgroups are covalently or non-covalently bonded to one or morebiomolecules. In one embodiment, the biomolecule is an antibody. In oneembodiment, the biomolecule is a peptide.

In one embodiment, the biomolecule is a nucleic acid. In one embodiment,the biomolecule is RNA. In one embodiment, the biomolecule is DNA.

In some aspects, one or more photoactive groups or compounds includebiomolecules that have one or more photoactive groups conjugated to it,i.e. covalently attached, much like a tag. In some aspects, the one ormore photoactive groups are not covalently attached to the biomoleculebut rather they are bound to it in a complex or by other means ofchemical bonding such as by an ionic bond, a Van der Waals bond, or ahydrogen bond.

In a general aspect, additional components such as a solvent, reagentssuch as homolysis initiating compounds, whole biological cells, and/oradditional photoactive compound(s) or biomolecules (e.g. additionalantibodies) may be present or otherwise added to the substrate and/orbiological molecule.

In one embodiment, the biomolecule is any combination of, or all of, oneor more peptides and/or one more nucleotides having up to twophotoactive groups on any given single peptide and/or nucleotide. In oneembodiment, the biomolecule is a cell receptor antigen.

Methods of Use

The technology described herein related to modified probes and relatedmethods, compositions, kits, and systems for binding probes modified tocomprise a photoactive group and specifically binding the probes totarget biomolecules for detection via monitoring pH after exposure ofthe probes to an activating radiation.

In some embodiments, provided herein is a method for detecting a targetbiomolecule, comprising: a) providing probe capable of bindingspecifically to a target biomolecule, wherein said probe is bound to aphotoacid generator or a photobase generator; b) contacting a samplesuspected of comprising said target biomolecule with said probe; c)removing unbound probes from said sample; d) exposing said sample to anwavelength of light capable of activating said photoacid generator orsaid photobase generator, such that said probe, if bound to said targetbiomolecule, releases an acid or a base upon exposure to said wavelengthof light; and e) detecting a concentration of ions in the sample,thereby identifying the presence or absence of said target analyte basedon a change of said concentration of ions.

In some embodiments, the probe comprises a polynucleotide or apolypeptide. In some embodiments, the probe is an antibody. In someembodiments, the concentration of ions is determined by measuring anionic strength of the sample. In some embodiments, the ionic strength ismeasured using an ion-sensitive field effect transistor. In someembodiments, the sample is immobilized on the surface of a substrate.

In some embodiments, the substrate is an array. In some embodiments, thearray comprises a plurality of wells, wherein said wells each comprise asensor for detecting an ionic strength of a solution in said wells. Insome embodiments, the sensor is an ion-sensitive field effecttransistor.

In some embodiments, provided herein is a method of detecting a sequenceidentity of a target polynucleotide, comprising: a) providing asubstrate an immobilized target polynucleotide hybridized to a primer orprobe; b) contacting said immobilized target polynucleotide with asolution comprising reagents for performing a polymerase extensionreaction, said solution comprising a set of modified nucleotidescomprising a photoactive group and a blocking group; c) exposing saidsubstrate to conditions to promote incorporation of one of said modifiednucleotides at the 3′ end of said primer or probe; d) washing saidsubstrate to remove unbound modified nucleotides; e) exposing saidimmobilized target polynucleotide to a wavelength of light to inducesaid photoactive group to generate an acid or a base, thereby generatinga detectable change in ion concentration in a solution surrounding saidimmobilized target polynucleotide if said modified nucleotide isincorporated into said target polynucleotide; f) detecting said changein ion concentration; and g) determining a sequence identity of saidtarget polynucleotide from said detected change in ion concentration.

The technology described herein relates to modified nucleotides andrelated methods, compositions, kits, and systems for sequencing nucleicacids. Sequencing by synthesis relies on the incorporation of anucleotide into a growing strand to form a correct cognate pair with atemplate strand. Either one nucleotide can be added at a time, and theincorporation of the correct nucleotide detected by various methods, ormultiple nucleotides can be added, and the identity of the incorporatednucleotide for each growing strand identified by a detectable marker onthe incorporated nucleotide after removal of all non-bound nucleotides.This type of reaction normally proceeds one nucleotide at a time, andthus the modified nucleotides additional comprise a removable blockinggroup, i.e., an element bound to the nucleotide that preventsincorporation of another nucleotide into the growing strand. This can beas simple as a dideoxy terminator, or can be a reversible terminator,allowing for subsequent polymerization of the growing strand foradditional sequencing detection.

In many instances of sequencing by synthesis, a fluorophore is used asthe detectable marker. However, imaging an array comprising millions offluorophores and processing that image into useable data can takesignificant time and is computationally intensive. Furthermore, the useof fluorophores as a detectable marker introduces limitations on theminimum size of features on an array due to diffraction-limited sensing.Electronic detection, such as ISFET, can remove the diffraction-limitedminimum size, and allow for simultaneous detection of a signal frommillions of samples on multiple arrays. However, limitations on theavailability of detectable markers to effectively distinguish betweenmultiple nucleotides using electronic sensing is limited, andsensitivity needs to be improved. Therefore, provided herein areimproved modified nucleotides to facilitate electronic detection todistinguish between at least four different nucleotides in a sequencingby synthesis reaction with high sensitivity and specificity.

The modified nucleotide provided herein can include photoactive groupscapable of generating an ionic signal when cleaved by light. In someembodiments, the photoactive groups are photoacid generators thatdecrease the pH of a solution when exposed to light. In someembodiments, the photoactive groups are photobase generators thatincrease the pH of a solution when exposed to light. Thus, according tomethods provided herein, a modified nucleotide comprising a photoactivegroup can be incorporated into a growing strand in a reaction chamber ifit forms a complementary base pairing with the template strand, followedby removal of un-incorporated nucleotides from the reaction chamber.Then, the reaction chamber can be exposed to light sufficient to inducephotocleavage of the photoactive group, and the pH of the solution canbe monitored to determine the effect of the exposure to light on thereaction solution. A change in pH and its magnitude as detected by ISFETcan be used to determine whether a nucleotide was incorporated, and ifso, the identity of this nucleotide. Thus, in some embodiments, providedherein are novel methods and compositions for sequencing using modifiednucleotides comprising photoactive groups capable of generating an ionicsignal, e.g., through the release of an acid or a base, such as aphotoacid or photobase generator.

In some embodiments, sequencing is performed by sequencing by synthesisof a clonal population of oligonucleotides, or by sequencing bysynthesis of a single molecule. In some embodiments, a signal isgenerated to determine the identity of a nucleotide incorporated intothe growing strand by electronic detection.

In previous generations of sequencing using electronic detection, onetype of nucleic base at time added for sequencing relays on weak H⁺produced by each base. This not only limits the throughput ofsequencing, but also requires a very sensitive Ion sensitive Fieldeffective transistor which limits the feature size of the ion sensingGATE of the transistor, and therefore the diversity of sequencingsignals that can be generated by different nucleotides.

Herein we provide improved methods and compositions for electronicdetection of nucleotide or base identity using sequencing by synthesis.Herein we provide modified nucleotides that do not rely on a weak H⁺produced by nucleic base addition, but instead can be detected by astrong H⁺ or OH⁻ produced by a non-interfering photo-energy to produce adesired ion strength for detection in a reaction area on an array. Thesecompositions allow us to decouple ion production for sensing from thenucleic acid base coupling to the growing strand. Since a strong H+ orOH− signal with varying ion strength can be generated by our modifiednucleotides, then four nucleic acid bases can be simultaneously added ateach coupling to sequence genome to generate a very high throughput.Furthermore, the strong electronic signal produced allows sensitivedetection and specific discrimination among the four nucleotides toenhance the accuracy of sequencing information generated.

In some embodiments, the modified nucleotides comprise photoactivegroups (i.e., photosensitive molecules) such as a photoacid or aphotobase generator (i.e., a molecule capable of generating aPhotogenerated acid or base), and can be referred to herein as aphotosensitive molecule (PM). In some embodiments, the photosensitivemolecule (PM) can be a molecule capable of producing a designed pHchange when exposed UV light. In some embodiments, the photogeneratedacid or base can be based on salt formulation, for example onephotoactive positive or negative charged molecule and one base or acidmolecule together form a salt. In some embodiments, the Photogeneratedacid or base can be based on photo-cleavage, for example the PMcomprises one photocleavage group (e.g., a linker) and one photoactivegroup covalently bonded to an acid or base which is released uponexposure to a light source.

Shown below is an example of a reaction of a nucleotide comprising aphotoactive group and a removable blocking group (indicated as a“STOPPER”) to control polymerization during sequencing by synthesis. Thephotoactive group is cleaved upon exposure to radiation to generate adetectable species.

In some embodiments, the modified nucleotides or photosensitivemolecules comprising photoactive groups comprise a nucleotide moleculebound to a photocleavable linker bound to a photoinduced base or acidproducing molecule. In some embodiments, the modified nucleotidesfurther comprise a removable blocking group to inhibit addition of morethan one nucleotide to the end of the growing strand. Examples ofremovable blocking groups include dideoxy terminators and reversibleterminators. The modified nucleotide may also include a photoactivegroup that also acts as a removable blocking group, such that, whennon-interfering photo-energy is applied to cleave photobase or photoacidgenerator, a signal for detection is generated and the growing strand isactivated to allow coupling of the next incoming nucleotide during thenext cycle of nucleotide addition.

Sequencing can occur on an array to template strands bound to an arrayin a chamber for electronic or ionic detection. Examples of methods ofelectronic or ionic detection include ISFET, ChemFET, and MOSFET. Anarray can comprise a plurality of reaction chambers or areas eachcapable of detecting ionic changes to the solution in each of thereaction chambers or areas. Each reaction chamber or area can comprise aplurality of clonal oligonucleotides for sequencing, or a single boundoligonucleotide for single molecule synthesis.

As provided herein, sequencing of oligonucleotides bound in an array cancontinue as follows: A mixture comprising reagents for amplification ofprimer extension, e.g., a polymerase, a set of modified nucleotides andother reagents to facilitate the incorporation of the correct nucleotideinto the growing strand hybridized to a template to be sequenced, isadded to a reaction chamber (FIG. 7). The reaction is allowed to proceedto incorporate one complementary modified nucleotide into the growingstrand. Then the reagents including unbound modified nucleotides areremoved from the solution, e.g., by washing, leaving only the modifiednucleotide (or nucleotides for clonal sequencing) incorporated into thegrowing strand in the reaction chamber. The reaction chamber is exposedto light (e.g., UV light) (FIG. 7), which activates the photobase orphotoacid generators to adjust the pH of the reaction chamber. Thischange in ionic conditions can then be detected by the sensors in thereaction chamber, e.g., ISFET. The pH of the chamber is detected and canbe used to determine the identity of the nucleotide incorporated intothe growing strand in the preceding nucleotide addition cycle, which canthen be used to determine the identity of the nucleotide base in thetemplate strand.

In some embodiments, as shown in FIG. 7, the reagents for amplificationinclude four modified nucleotides. These four modified nucleotides areeach capable of adjusting the pH of the solution in the reaction chamberby a defined amount to provide information on the identity of theincorporated nucleotide. In some embodiments, as shown in FIG. 7, thefour modified nucleotides can comprise photobase or photoacidgenerators. Two or more photobase generators can be used which havedistinguishable ionic strengths to allow discrimination by unique pHlevels in the reaction chamber after exposure to light. Two or morephotoacid generators can be used which have distinguishable ionicstrengths to allow discrimination by unique pH levels in the reactionchamber after exposure to light.

During the photoacid or photobase production, the ion level changes inthe reaction chamber solution generated by the photoacid/photobasegeneration and detected by ion-sensitive gate of field effectivetransistor can then be classified and analyzed according to the fourtypes of nucleic acid bases (FIG. 8). In some embodiments, additionalcycles of sequencing are perform to generate sequencing information fora stretch of oligonucleotides on the template strand. In thisembodiment, the reaction chamber can be neutralized to revert the pH to7 before proceeding with the next reaction. In addition, if a removableblocking group is still present on the modified nucleotide, it should beremoved to allow addition of the next incoming modified nucleotide onthe growing strand in the next cycle. Multiple cycles can be performedand analyzed as shown in FIG. 8 to detect a sequence ofoligonucleotides.

Arrays

Methods of detection using photoactivated compounds is described hereinusing arrays. The arrays comprise multiple pH sensors, such asion-sensitive field effect transistors (ISFET), which are sensitive tosmall perturbances in ionic strength and can be provided in miniaturizedfeatures on an array. Shown in FIG. 9 is an example of an array of wellseach comprising a pH sensor, such as an ISFET sensor. Each well can haveone or more target biomolecules bound therein, which can be detected orotherwise characterized, such as by sequencing, using the methods andphotoactive compounds as described herein. Uses of the arrays disclosedherein can include research applications, therapeutic purposes, medicaldiagnostics, and/or stratifying one or more patients.

Any of the arrays described herein can be used as a research tool or ina research application. In one aspect, arrays can be used for highthroughput screening assays. For example, substrates comprisingimmobilized probes comprising DNA (deoxyribonucleic acid), RNA(ribonucleic acid), PNA (peptide nucleic acid), LNA (locked nucleicacid), or hybrid combinations thereof can be tested by subjecting thearray to a DNA or RNA molecule and identifying the presence or absenceof the complimentary DNA, RNA, or PNA molecule, e.g., by detecting atleast one change among the features of the array. PNA-DNA chimericsubstrates can be tested by subjecting the array to a complementary DNAmolecule and performing a single nucleotide extension reaction todetermine whether the substrate is biologically active, and to identifya SNP in a sample.

In some embodiments, an array can be used for detection of sequencevariants in a sample, e.g., single nucleotide polymorphisms (SNPs).Detection of sequence variants can occur through observingsequence-specific hybridization of labeled molecules to a probe on anarray. Detection of sequence variants can also occur through binding ofa sequence suspected of having a sequence variant to a probe on anarray, followed by performing a polymerase extension reaction with alabelled nucleotides. In preferred embodiments, PNA-DNA chimericoligonucleotide probes are bound to the array and hybridize tonucleotide sequences from a sample suspected of comprising a sequencevariant. The PNA-DNA chimeric oligonucleotides are enzymatically active,i.e., they are capable of acting as a substrate for complementarynucleotide incorporation into a growing strand using a polymerase underpreferred conditions for polymerization. FIG. 10 provides an exemplaryscheme for detecting the identity of a sample oligonucleotide hybridizedto a PNA-DNA chimeric oligonucleotide covalently attached to the arrayusing a polymerase-based single nucleotide extension reaction with alabeled nucleotide. Examples of PNA-DNA chimeric oligonucleotide-basedmethods for SNP detection are provided in U.S. Pat. No. 6,316,230,incorporated herein by reference in its entirety.

Arrays can also be used in screening assays for ligand binding, todetermine substrate specificity, or for the identification ofcomplimentary DNA, RNA, PNA molecule that are expressed in certain cellsin vivo or in vitro. Labeling techniques, protease assays, as well asbinding assays useful for carrying out these methodologies are generallywell-known to one of skill in the art.

In some embodiments, an array can be used to represent a predefined PNAchain as a sequence of overlapping PNA sequences. For example, the PNAsequence of a known gene is divided into overlapping sequence segmentsof any length and of any suitable overlapping frame, and PNA chainscorresponding to the respective sequence segments are in-situsynthesized as disclosed herein. The individual PNA segments sosynthesized can be arranged starting from the amino terminus of thepredefined PNA chain.

In some embodiments, a sample is applied to an array having a pluralityof random PNA chains. The random PNA chains can be screened and BLASTedto determine homologous domains with, e.g., a 90% or more identity to agiven nucleotide sequence. In some aspect, the whole PNA sequence canthen be synthesized and used to identify potential markers and/or causesof a disease of interest.

In some embodiments, an array is used for high throughput screening ofone or more genetic factors. DNA or RNA expression associated with agene can be investigated through PNA hybridization, which can then beused to estimate the relation between gene and a disease.

In another example, an array can be used to identify one or morebiomarkers. Biomarkers can be used for the diagnosis, prognosis,treatment, and management of diseases. Biomarkers may be expressed, orabsent, or at a different level in an individual, depending on thedisease condition, stage of the disease, and response to diseasetreatment. Biomarkers can be, e.g., DNA, RNA, PNA, proteins (e.g.,enzymes such as kinases), sugars, salts, fats, lipids, or ions.

Arrays can also be used for therapeutic purposes, e.g., identifying oneor more bioactive agents. A method for identifying a bioactive agent cancomprise applying a plurality of test compounds to an array andidentifying at least one test compound as a bioactive agent. The testcompounds can be small molecules, aptamers, oligonucleotides, chemicals,natural extracts, peptides, proteins, fragments of antibodies, antibodylike molecules, or antibodies. In some embodiments, test compounds arehybridizing DNA, RNA or PNA sequences. The bioactive agent can be atherapeutic agent or modifier of therapeutic targets. Therapeutictargets can include phosphatases, proteases, ligases, signaltransduction molecules, transcription factors, protein transporters,protein sorters, cell surface receptors, secreted factors, andcytoskeleton proteins.

In one aspect, also provided are arrays for use in medical diagnostics.An array can be used to determine a response to administration of drugsor vaccines. For example, an individual's response to a vaccine can bedetermined by detecting the gene expression levels of the individual byusing an array with PNA chains or PNA-DNA chimeric oligonucleotidechains representing particular genes associated with the induced immuneresponse. Another diagnostic use is to test an individual for thepresence of biomarkers, wherein samples are taken from a subject and thesample is tested for the presence of one or more biomarkers.

Arrays can also be used to stratify patient populations based upon thepresence or absence of a biomarker that indicates the likelihood asubject will respond to a therapeutic treatment. The arrays can be usedto identify known biomarkers to determine the appropriate treatmentgroup. For example, a sample from a subject with a condition can beapplied to an array. Binding to the array may indicate the presence of abiomarker for a condition. Previous studies may indicate that thebiomarker is associated with a positive outcome following a treatment,whereas absence of the biomarker is associated with a negative orneutral outcome following a treatment. Because the patient has thebiomarker, a health care professional may stratify the patient into agroup that receives the treatment.

In some embodiments, a method of detecting the presence or absence of aexpressed gene of interest in a sample can include obtaining an arraydisclosed herein and contacted with a sample suspected of comprising theDNA or RNA sequence of a gene of interest; and determining whether thegene of interest is expressed in the sample by detecting the presence orabsence of binding to one or more features of the array. In someembodiments, the DNA or RNA sequence of the gene of interest can beobtained from a bodily fluid, such as amniotic fluid, aqueous humour,vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid,cerumen, chyle, endolymph, perilymph, feces, female ejaculate, gastricacid, gastric juice, lymph, mucus, peritoneal fluid, pleural fluid, pus,saliva, sebum, semen, sweat, synovial fluid, tears, vaginal secretion,vomit, or urine.

In some embodiments, a method of identifying a vaccine candidate caninclude obtaining an array disclosed herein contacted with a samplederived from a subject previously administered the vaccine candidate,wherein the sample comprises a plurality of DNA or RNA sequences; anddetermining the binding specificity of the plurality of DNA or RNAsequences to one or more features of the array. In some embodiments, thefeatures comprise a plurality of distinct, nested, overlapping PNAchains comprising subsequences derived from a known nucleotide sequence.

In one embodiment, the two or more different photoactive groups arebound by the target biomolecules on the array or substrate such thatupon exposure to light, one group reacts and affects the pH selectively.In such embodiments, both of, or the plurality of, photoactive groupswill affect the pH, however, one photoactive group will affect the pHsignificantly more. In some such embodiments, one photoactive group willaffect the pH at least half an order of magnitude more, viz. ameasurable change in the pH of the surrounding environment is at least0.5 units in magnitude different than what is predicted for theplurality of one or more photoactive groups to affect the pH. In somesuch embodiments, one photoactive group will affect the pH at least oneorder of magnitude more. In some such embodiments, one photoactive groupwill affect the pH at least two orders of magnitude more. In some suchembodiments, two or more photoactive groups will each affect the pH atleast half an order of magnitude more than what is predicted for eachone of the other one or more photoactive groups to affect the pH.

Herein also disclosed the inventors have contemplated that each andevery embodiment and/or aspect may be used in any combination with anyother embodiment and/or aspect. For examples, photoactive compounds orfunctional groups of the present disclosure can include any combinationof the HOMO. LUMO, pKa, or absorbed light energy within the ranges setforth.

Substrates and arrays suitable for binding probes and targetbiomolecules for use in detection of the photoactive groups and targetbiomolecules as described herein are disclosed at least in PCTPublication No. WO 2013/119845, PCT Publication No. WO 2014/052989, PCTPublication No. WO 2014/078606, PCT Publication No. WO 2014/127328, PCTPublication No. WO 2015/127409, PCT Publication No. WO 2016/145434, andPCT Publication No. WO 2017/117292, the entirety of which are eachincorporated by reference.

Results Analysis and Information Storage

In some embodiments, the method of sequencing using modified nucleotidesor the array comprising hybrid PNA/DNA probe oligonucleotides is part ofa system, such as a diagnostic or sequencing system or platform thatprovides highly-multiplexed sequencing results to users.

In some embodiments, provided herein is a sequencing system in which asample is added to a sequencing array, and oligonucleotides from thesample are sequenced using the methods and compositions describedherein. FIGS. 11 and 12 provide examples of systems that can be used tomount an array provided herein and receive electrical signals from eachwell to perform a sequencing reaction. FIG. 11 shows a diagram of asystem including a device that interfaces with a chip or array asdescribed herein to collect data from the chip and process it. Examplesof mechanisms to obtain information from each of the multiple reactionareas on the array are shown. FIG. 12 shows a plate base contact toplate base readout stage and elements for UV exposure to inducephotoactivation or photocleavage, according to an embodiment of theinvention.

A reader can be used to read the sequencing array. For example, thesequencing array may be inserted into the reader, or placed on or insidethe reader. In some embodiments, the reader provides an interface withthe sequencing array to allow multiplexed electronic detection ofsequencing, e.g., by detection of changes to pH such as through ISFETdetection. The reader identifies electrical signals in each reactionarea or well on an array from incorporated modified nucleotides afterexposure to light to induce photoacid or photobase generation. Thereader then reads the pH of each reaction chamber, and determines theidentity of the incorporated nucleotide. In some embodiments, the readerincludes a display, such as a screen, that allows it to display to theuser the results of sequencing. In some embodiments, the confidencelevel of a nucleotide identification can also be determined and providedfor display on the reader.

In some embodiments, a software program installed on the reader maycompare one or more electronic signals from a reaction chamber with areference electronic signal associated with a nucleotide. The programcan perform this process through a number of cycles to generate asequence of an oligonucleotide contained within a reaction area of thearray. In some embodiments, the software program is a computer readablemedium storing instructions on the reader that when executed by aprocessor within the reader cause the processor to perform certainactions, such as identifying the pH of a reaction area or storingcertain data, such as sequencing data. In some embodiments, the softwareprogram comprises one or more software modules that perform each of thevarious functions described above for the reader.

FIG. 13 illustrates a system environment 101 including the sequencingsystem 100 described above, according to an embodiment. The systemenvironment 101 can further include one or more client devices 110, oneor more servers 130, a database 105 accessible to the server 130, whereall of these parties are connected through a network 120. In otherembodiments, different and/or additional entities can be included in thesystem environment 101.

The system environment 101 allows the results from the reader 140 to beshared via network 120 with one or more other users at their clientdevices 110, including being shared with family, friends, physicians orother medical personnel, schools, civil response teams, among others.Results can also be uploaded to the web.

The network 120 facilitates communications between the components of thesystem environment 101. The network 120 may be any wired or wirelesslocal area network (LAN) and/or wide area network (WAN), such as anintranet, an extranet, or the Internet. In various embodiments, thenetwork 120 uses standard communication technologies and/or protocols.Examples of technologies used by the network 120 include Ethernet,802.11, 3G, 4G, 802.16, or any other suitable communication technology.The network 120 may use wireless, wired, or a combination of wirelessand wired communication technologies. Examples of networking protocolsused for communicating via the network 120 include multiprotocol labelswitching (MPLS), transmission control protocol/Internet protocol(TCP/IP), hypertext transport protocol (HTTP), simple mail transferprotocol (SMTP), and file transfer protocol (FTP). Data exchanged overthe network 120 may be represented using any suitable format, such ashypertext markup language (HTML) or extensible markup language (XML). Insome embodiments, all or some of the communication links of the network120 may be encrypted using any suitable technique or techniques.

The client device(s) 110 are computing devices capable of receiving userinput as well as transmitting and/or receiving data via the network 120.In one embodiment, a client device 110 is a conventional computersystem, such as a desktop or laptop computer. Alternatively, a clientdevice 110 may be a device having computer functionality, such as apersonal digital assistant (PDA), a mobile telephone, a smartphone oranother suitable device. A client device 110 is configured tocommunicate via the network 120.

In some embodiments, the system environment 101 may include one or moreservers, for example where the sequencing system includes a service thatis managed by an entity that communicates via the network 120 with thereader 140 and/or any of the client devices 110. The server 130 canstore data in database 105 and can access stored data in database 105.Database 105 may be an external database storing sequencing data,medical information, user or patient history data, etc. The server 130may also store data in the cloud. In some embodiments, the server 130may occasionally push updates to the reader 140, or may receive resultdata from the reader 140 and perform certain analyses on that resultdata and provide the analyzed data back to the reader 140 or to a clientdevice 110.

In some embodiments, the reader 140 functionality can be included in aclient device 110, such as a mobile phone, and can be operated via amobile application installed on the phone. In these embodiments, adevice may be attached to the phone that allows the phone to read thetest strip, or the phone's own internal hardware (e.g., imaginghardware) can be used to read the test strip. The mobile applicationstored on the phone can process the results read from the test strip andshare the results with other devices 110 on the network 120.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1A: Synthesis of a PNA-DNA Probes on an Array Synthesis of PNASequence

To generate a PNA-DNA chimera on the surface of an array, a PNA oligo ofa desired sequence at a specific site was first synthesized on a chipaccording to the protocol provided below:

A location-specific PNA sequence synthesis is performed on an array asfollows: A wafer is spin-coated at 2000-4000 rpm (preferably 2500-3000rpm) for 10-180 seconds (preferably for 60-120 seconds) with aphotoresist composition comprising a photobase generator as describedabove. The wafer and photoresist is exposed to 248 nm ultraviolet lightin a deep ultra violet scanner tool according to a pattern defined by aphotomask, wherein the locations exposed to ultraviolet light undergobase generation due to the presence of a photobase generator in thephotoactive coupling solution in the photoresist. The expose energy canbe from 1 mJ/cm² to 100 mJ/cm² (preferably 30-60 mJ/cm²).

After UV exposure, the surface of the wafer is post baked in a bakemodule. The post bake temperature can vary between 75° C. to 115° C.,for a duration of at least 60 seconds (but not usually exceeding 180seconds). The base generated at the UV-exposed regions removes aprotection group on the amino groups. The photoresist is then stripped.

The free amino group is then coupled to activated R2-acetic acid by spincoating R2-acetic acid with activation agents and reacted in a bakemodule with the temperature varying from 55° C. to 115° C. for 60-240seconds. Excess R2-acetic acid is removed.

Following addition of activated R2-acetic acid, a displacement reactionwith a mono-amino protected ethylenediamine is performed by addition ofa displacement mixture onto the surface of the wafer. This displacementmixture is spin coated on the wafer and reacted in a bake module withthe temperature varying from 55° C. to 115° C. for 30 seconds-300seconds (preferably 120 seconds). In some embodiments, a mono-aminoprotection group can be any amino protection group as mentioned earlier.

Next, a coupling reaction is performed with the peptide nucleic acidmonomer acetic acid. The displaced amine is coupled to the activated PNAmonomer acetic acid by spin coating the activated PNA monomer aceticacid with activation agents and allowing to react for 90-300 seconds.

Optionally, a capping solution coat is applied on the surface to preventany non-reacted amino groups on the substrate from reacting to the nextcoupling molecule. The capping solution includes a solvent, a polymer,and a coupling molecule. The capping solution is spin-coated on thewafer at 1500-3500 rpm for at least 30 seconds and reacted in a bakemodule with the temperature varying from 55° C. to 95° C. for 30seconds-90 seconds (preferably 60 seconds) to complete one cycle.

This entire cycle can be repeated as desired with different nucleic acidmonomers each time to obtain desired PNA sequences at specific sites onan array.

Part 2—Synthesis of PNA-DNA Chimera Step.

Addition of a sequence-specific DNA nucleotide to the end of a PNAsequence at specific locations on an array is performed as follows: Thewafer comprising PNA sequences is spin-coated at 2000-4000 rpm(preferable 2500-3000 rpm) for 10-180 seconds preferably for 60-120seconds with the photoresist and is exposed to 248 nm ultraviolet lightin a deep ultra violet scanner tool according to a pattern defined by aphotomask, wherein the locations exposed to ultraviolet light undergobase generation due to the presence of a photobase generator in thephotoactive coupling solution in the photoresist. The expose energy canbe from 1 mJ/cm² to 100 mJ/cm² (preferably 30-60 mJ/cm²).

After UV exposure, the surface of the wafer is post baked in a bakemodule. The post bake temperature can vary between 75° C. to 115° C.,for a duration of at least 60 seconds (but not usually exceeding 180seconds). The generated base deprotects the protection of amino groupsof the PNA sequences in the exposed regions. The photoresist is thenstripped.

Reverse DNA amidites are activated with phosphoramidite activationsolution (including e.g., a tetrazole catalyst) and then coupled to thefree amine.

Optionally, a capping solution coat is applied on the surface to preventany non-reacted amino groups on the substrate from reacting to the nextcoupling molecule. The capping solution includes a solvent, a polymer,and a coupling molecule. The capping solution is spin-coated on thewafer at 1500-3500 rpm for at least 30 seconds and reacted in a bakemodule with the temperature varying from 55° C. to 95° C. for 30seconds-90 seconds (preferably 60 seconds) to complete one cycle. Thephosphite-triester formed in the coupling step is then converted to astable form which is achieved by iodine oxidation in the presence ofwater and pyridine.

Part 3—Reverse DNA oligonucleotide synthesis.

A location-specific DNA sequence reverse synthesis (5′ to 3′) isperformed at the end of each PNA-DNA chimera on the array as follows: Awafer is spin-coated at 2000-4000 rpm (preferably 2500-3000 rpm) for10-180 seconds (preferably for 60-120 seconds) with a photoresistcomposition comprising a photobase generator as described above. Thewafer and photoresist is exposed to 248 nm ultraviolet light in a deepultra violet scanner tool according to a pattern defined by a photomask,wherein the locations exposed to ultraviolet light undergo acidgeneration due to the presence of a photoacid generator in thephotoactive coupling solution. The expose energy can be from 1 mJ/cm² to100 mJ/cm² (preferably 30-60 mJ/cm²).

After UV exposure, the surface of the wafer is post baked in a bakemodule. The post bake temperature can vary between 75° C. to 115° C.,for a duration of at least 60 seconds (but not usually exceeding 180seconds). The acid generated deprotects the protection of hydroxylgroups of the 3′-end of DNA sequence in the exposed regions. Thephotoresist is then stripped.

Reverse DNA amidites are activated with phosphoramidite activationsolution (including e.g., a tetrazole catalyst) and then coupled to thefree hydroxyl group.

Optionally, a capping solution coat is applied on the surface to preventany non-reacted amino groups on the substrate from reacting to the nextcoupling molecule. The capping solution includes a solvent, a polymer,and a coupling molecule. The capping solution is spin-coated on thewafer at 1500-3500 rpm for at least 30 seconds and reacted in a bakemodule with the temperature varying from 55° C. to 95° C. for 30seconds-90 seconds (preferably 60 seconds) to complete one cycle. Thephosphite-triester formed in the coupling step is then converted to astable form which is achieved by iodine oxidation in the presence ofwater and pyridine.

Example 2: Hybridization of PNA-DNA Chimera to Oligonucleotide DNASequence and Extension with Polymerase

To determine the biological activity of a PNA-DNA chimera sequencesynthesized per the above protocol, a 34-mer length PNA-DNA chimerasequence 5′-GTGGAAATTTGACATAGTCTCAGATGCCTAT(TAT)-3′ (SEQ ID NO: 7) wassynthesized according to Example 3 with (TAT) being the DNA oligomerportion of the PNA-DNA chimera. Four oligonucleotide DNA sequencescomplimentary to the sequence of the PNA-DNA chimera with one additionalnucleotide were synthesized (IDT).

S1: (SEQ ID NO: 8) CACCTTTAAACTGTATCAGAGTCTACGGATAATAa S2:(SEQ ID NO: 9) CACCTTTAAACTGTATCAGAGTCTACGGATAATAc S3: (SEQ ID NO: 10)CACCTTTAAACTGTATCAGAGTCTACGGATAATAg S4: (SEQ ID NO: 11)CACCTTTAAACTGTATCAGAGTCTACGGATAATAt

A primer extension reaction to detect incorporation of the correctcomplementary nucleotide in a primer extension reaction was performed.Alexa 405 ddATP, Alexa 488 ddCTP, Alexa 555 ddGTP, Alexa 647 ddTTP weresynthesized by techniques well known to one skilled in the art.Hybridization and polymerase extension on each of the 4 chips wereperformed as follows. Oligomers S1, S2, S3 and S4 were diluted 1:1000(100 nM) in 1×DNA Polymerase Buffer (Clontech), 20 nmol of MgCl2, 1 unitTitanium Taq DNA Polymerase (Clontech), and all 4 labelled ddNTPslabelled monomers (each 25 pmoles). Hybridization was done in ahybridization chamber at 55° C. for 30 minutes followed by washing thechips in 0.1× Ssarc buffer at 40° C. for 5 minutes twice followed byrinsing in DI Water. The chip was then scanned on a Nikon A1R confocalmicroscope which included the 4 wavelengths of the dyes used in theddNTPs and results are depicted in Table 1.

TABLE 1 Hybridization and Extension of PNA-DNA Chimera Sequence Sequence405 nm 488 nm 561 nm 640 nm S1 (a) 950.02 875.54 1047.8 65124.23 S2 (c)1051.3 954.2 65531.25 875.6 S3 (g) 800.3 65014.78 802.5 1068.9 S4 (t)65121.13 1012.98 946.6 780.9

Example 3: Genotyping Using PNA Sequence Hybridization

Testing of the PNA synthesis for a genotyping SNP-based application wasperformed. Genotyping of MTHFR region, with the well-known mutations,C677T and A1298C, were tested using 20 DNA samples. The DNA samples hada known genotyping result which were determined using Real-Time PCR.

PNA Sequences were as follows:

(SEQ ID NO: 12) GGAGAAGGTGTCTGCGGGAG(C)CGATTTCATCATCACGCAGC,(SEQ ID NO: 13) GGAGAAGGTGTCTGCGGGAG(T)CGATTTCATCATCACGCAGC,(SEQ ID NO: 14) GGAGGAGCTGACCAGTGAAG(A)AAGTGTCTTTGAAGTCTTCG,(SEQ ID NO: 15) GGAGGAGCTGACCAGTGAAG(C)AAGTGTCTTTGAAGTCTTCG.

PNA sequences were synthesized on a chip using the methods given above.The location of the SNP is indicated in ( ) region (surrounded byparentheses) and is synthesized in the middle of the sequence.

DNA were extracted from the samples (buccal swabs) using methods knownto one skilled in the art. A standard PCR reaction using forward primerand biotin-labelled reverse primers was performed on the extracted DNAsamples. Hybridization of the PCR product on the chip was performed withthe PCR product (20 ul) diluted in hybridization buffer 0.1× SsarcBuffer 60 mM sodium chloride (Sigma) (80 ul), 6 mM sodium citrate(Sigma), 0.72 weight % N-lauroylsarcosine sodium salt solution (Sigma).Hybridization was done in a hybridization chamber at 55° C. for 2 hoursfollowed by washing the chips in 0.1× Ssarc buffer 40° C. for 5 minutestwice. This was followed by an incubation with 1 ng/ml Atto 488Streptavidin (Rockland) diluted in PBS buffer, washing the chips in PBSBuffer twice and rinsing in DI Water. The chip was then scanned on aNikon A1R confocal microscope and results are depicted in Table 2 andTable 3.

TABLE 2 677C > T Mutation Results (PNA) SEQ ID NO: SEQ ID NO: CalculatedSample ID Original Result 12 (C) 13 (T) Ratio (C/T) Result MT1Homozygous 65521.21 18343.16 3.571969606 Homozygous Wild C/C Wild C/CMT2 Homozygous 65227.42 17390.41 3.750769533 Homozygous Wild C/C WildC/C MT3 Homozygous 65093.45 18262.83 3.564258661 Homozygous Wild C/CWild C/C MT4 Homozygous 65386.72 16498.11 3.963285491 Homozygous WildC/C Wild C/C MT5 Homozygous 65245.82 17957.35 3.633376862 HomozygousWild C/C Wild C/C MT6 Homozygous 65399.12 15087.35 4.334698937Homozygous Wild C/C Wild C/C MT7 Homozygous 65408.94 16166.174.046038115 Homozygous Wild C/C Wild C/C MT8 Heterozygous 32656.7329003.56 1.125955917 Heterozygous C/T C/T MT9 Heterozygous 31140.428860.93 1.078981169 Heterozygous C/T C/T MT10 Heterozygous 30317.0927075.3 1.119732376 Heterozygous C/T C/T MT11 Heterozygous 29953.0530101.53 0.99506736 Heterozygous C/T C/T MT12 Heterozygous 34884.6232957.31 1.058478984 Heterozygous C/T C/T MT13 Heterozygous 28468.4330134.87 0.944700608 Heterozygous C/T C/T MT14 Heterozygous 29909.2626689.12 1.12065366 Heterozygous C/T C/T MT15 Homozygous 16202.9465103.75 0.248878751 Homozygous Mutant T/T Mutant T/T MT16 Homozygous16759.95 65465.38 0.256012415 Homozygous Mutant T/T Mutant T/T MT17Homozygous 18019.34 65327.52 0.275830768 Homozygous Mutant T/T MutantT/T MT18 Homozygous 15153.13 65116.35 0.232708529 Homozygous Mutant T/TMutant T/T MT19 Homozygous 16837.25 65198.8 0.258244784 HomozygousMutant T/T Mutant T/T MT20 Homozygous 16315.66 65431.64 0.249354288Homozygous Mutant T/T Mutant T/T No No Template 931.04 1010.87 NoTemplate Template Control Control

TABLE 3 1298A > C Mutation Results (PNA) SEQ ID NO: SEQ ID NO:Calculated Sample ID Original Result 14 (A) 15 (C) Ratio (A/C) ResultMT1 Homozygous 65379.46 18466.16 3.540501111 Homozygous Wild A/A WildC/C MT2 Homozygous 65471.94 15279.46 4.284964259 Homozygous Wild A/AWild C/C MT3 Homozygous 65031.65 16581.28 3.92199215 Homozygous Wild A/AWild C/C MT4 Homozygous 65219.55 17032.97 3.829018075 Homozygous WildA/A Wild C/C MT5 Homozygous 65122.87 15781.57 4.126514029 HomozygousWild A/A Wild C/C MT6 Homozygous 65211.42 16471.62 3.959016782Homozygous Wild A/A Wild C/C MT7 Homozygous 65284.01 16098.4 4.055310466Homozygous Wild A/A Wild C/C MT8 Heterozygous 25864.99 26219.090.986494573 Heterozygous A/C C/T MT9 Heterozygous 27535.6 33971.80.810542862 Heterozygous A/C C/T MT10 Heterozygous 29798.31 25928.751.149238201 Heterozygous A/C C/T MT11 Heterozygous 31306.14 30412.041.02939954 Heterozygous A/C C/T MT12 Heterozygous 34735.37 26137.171.328964459 Heterozygous A/C C/T MT13 Heterozygous 34293.84 323931.058680579 Heterozygous A/C C/T MT14 Heterozygous 34029.48 29563.141.151077998 Heterozygous A/C C/T MT15 Homozygous 17863.25 65072.670.274512326 Homozygous Mutant C/C Mutant T/T MT16 Homozygous 15361.4665400.42 0.234883201 Homozygous Mutant C/C Mutant T/T MT17 Homozygous16092.89 65285.28 0.246501049 Homozygous Mutant C/C Mutant T/T MT18Homozygous 16038.93 65184.73 0.246053485 Homozygous Mutant C/C MutantT/T MT19 Homozygous 16752.2 65398.01 0.256157641 Homozygous Mutant C/CMutant T/T MT20 Homozygous 16298.91 65253.44 0.249778556 HomozygousMutant C/C Mutant T/T No No Template 931.04 1010.87 No Template TemplateControl Control

In this method, the SNP location is ideally close to the center of thesequence synthesized.

Example 4: Genotyping Using PNA-DNA Chimera and Primer Extension

Testing of the PNA-DNA chimera for a genotyping SNP-based applicationwas performed. Genotyping of MTHFR region, with the well-knownmutations, C677T and A1298C, were tested using 20 DNA samples. The DNAsamples had a known genotyping result which were determined usingReal-Time PCR. 34-mer length PNA-DNA chimera primer sequences5′-CTGAAGCACTTGAAGGAGAAGGTGTCTGCGG(GAG)-3′ (SEQ ID NO: 16) for the677C>T mutation and 5′-CTGAAGATGTGGGGGGAGGAGCTGACCAGTG(AAG)-3′ (SEQ IDNO: 17) for the 1298A>C mutation were synthesized according to themethods given above (with DNA nucleotide portion of the PNA-DNA chimeraenclosed in parentheses).

DNA were extracted from the samples (buccal swabs) using methods knownto one skilled in the art. A standard PCR reaction using forward andreverse primers was performed on the extracted DNA samples.Hybridization and polymerase extension on each of chips were performedas follows. The PCR product was mixed in 1×DNA Polymerase Buffer(Clontech), 20 nmol of MgCl₂, 1 unit Titanium Taq DNA Polymerase(Clontech), and all 4 labelled ddNTPs labelled monomers (each at 25pmol). Hybridization was done in a hybridization chamber at 55° C. for30 minutes followed by washing the chips in 0.1× Ssarc buffer 40° C. for5 minutes twice followed by rinsing in DI Water. The chip was thenscanned on a Nikon A1R confocal microscope which included the 4wavelengths of the dyes used in the ddNTPs and results are depicted inTable 4 and Table 5.

TABLE 4 677C > T Mutation Results (PNA-DNA Chimera) Original 405 nm 488nm 561 nm 640 nm Ratio Calculated Sample ID Result (A) (C) (G) (T) (C/T)Result MT1 Homozygous 987.88 65381.38 1068.03 1022.91 63.9170406Homozygous Wild C/C Wild C/C MT2 Homozygous 1079.22 65379.69 1003.711088.7 60.0529898 Homozygous Wild C/C Wild C/C MT3 Homozygous 890.1565426.33 1054.07 984.99 66.4233444 Homozygous Wild C/C Wild C/C MT4Homozygous 1019.28 65082.83 1072.37 1005.67 64.7158909 Homozygous WildC/C Wild C/C MT5 Homozygous 1054.92 65520.82 885.87 919.46 71.26010919Homozygous Wild C/C Wild C/C MT6 Homozygous 856.58 65086.76 1063 1081.4660.18415845 Homozygous Wild C/C Wild C/C MT7 Homozygous 1014.84 65048.371012.61 1020.77 63.72480578 Homozygous Wild C/C Wild C/C MT8Heterozygous 1086.58 28940.21 916.77 25640.7 1.128682524 HeterozygousC/T C/T MT9 Heterozygous 1045.85 34602.61 903.71 33874.1 1.021506402Heterozygous C/T C/T MT10 Heterozygous 920.14 30597.21 1085.44 32891.410.930249266 Heterozygous C/T C/T MT11 Heterozygous 1065.78 26105.81883.95 25875.75 1.00889095 Heterozygous C/T C/T MT12 Heterozygous1087.05 28716.64 1028.78 30765.41 0.933406706 Heterozygous C/T C/T MT13Heterozygous 1096.23 30296.19 1097.06 29260.09 1.035410007 HeterozygousC/T C/T MT14 Heterozygous 999.32 31152.26 1083.28 28772.12 1.082723831Heterozygous C/T C/T MT15 Homozygous 959.46 920.49 983.83 65264.850.014103917 Homozygous Mutant T/T Mutant T/T MT16 Homozygous 999.031017.52 1098.91 65228.95 0.015599209 Homozygous Mutant T/T Mutant T/TMT17 Homozygous 908.97 1031.03 1083.42 65170.19 0.015820577 HomozygousMutant T/T Mutant T/T MT18 Homozygous 1053.12 1000.45 961.54 65066.090.015375905 Homozygous Mutant T/T Mutant T/T MT19 Homozygous 1070.321075.46 1009.19 65326.99 0.016462721 Homozygous Mutant T/T Mutant T/TMT20 Homozygous 1059.73 915.48 854.63 65287.66 0.014022252 HomozygousMutant T/T Mutant T/T No No Template 1005.16 931.04 1018.61 1010.87 NoTemplate Template Control Control

TABLE 5 1298 A > C Mutation Results (PNA-DNA Chimera) Original 405 nm488 nm 561 nm 640 nm Ratio Calculated Sample ID Result (A) (C) (G) (T)(A/C) Result MT1 Homozygous 65488.6 860.42 898.56 1053.57 76.11236373Homozygous Wild A/A Wild A/A MT2 Homozygous 65344.12 942.87 1005.5859.47 69.30342465 Homozygous Wild A/A Wild A/A MT3 Homozygous 65439.64943 1040.81 1048.08 69.39516437 Homozygous Wild A/A Wild A/A MT4Homozygous 65352.13 1015.9 850.32 978.43 64.32929422 Homozygous Wild A/AWild A/A MT5 Homozygous 65258.17 1047.97 999.32 858.43 62.27102875Homozygous Wild A/A Wild A/A MT6 Homozygous 65327.7 871.43 984.33 971.4274.96609022 Homozygous Wild A/A Wild A/A MT7 Homozygous 65222.79 1032.191093.43 894.49 63.18874432 Homozygous Wild A/A Wild A/A MT8 Heterozygous32437.6 29322.89 851.72 897.02 1.106221113 Heterozygous A/C A/C MT9Heterozygous 28901.78 32234.2 972.78 930.13 0.896618498 Heterozygous A/CA/C MT10 Heterozygous 31930.24 34009.27 902.33 1072.67 0.938868726Heterozygous A/C A/C MT11 Heterozygous 31187.77 33959.45 1066.13 858.620.918382659 Heterozygous A/C A/C MT12 Heterozygous 35160.88 34129.731085.9 978.16 1.030212662 Heterozygous A/C A/C MT13 Heterozygous 26032.434987.94 980.73 1047.35 0.744039232 Heterozygous A/C A/C MT14Heterozygous 32902.79 33518.34 1024.53 998.06 0.981635427 HeterozygousA/C A/C MT15 Homozygous 853.23 65405.7 866.06 1096.1 0.013045193Homozygous Mutant C/C Mutant C/C MT16 Homozygous 861.56 65469.41 1045.551093.83 0.013159734 Homozygous Mutant C/C Mutant C/C MT17 Homozygous948.8 65335.08 1094.53 1025.03 0.014522061 Homozygous Mutant C/C MutantC/C MT18 Homozygous 853.16 65190.22 964.72 901.27 0.013087239 HomozygousMutant C/C Mutant C/C MT19 Homozygous 1014.49 65003.94 1015.66 922.790.015606592 Homozygous Mutant C/C Mutant C/C MT20 Homozygous 926.3265437.85 945.85 1063.8 0.014155722 Homozygous Mutant C/C Mutant C/C NoNo Template 1043.98 1043.85 986.73 1073.34 No Template Template ControlControl

In this method, the sequence synthesized on the chip contains the regionjust before the SNP location, thereby enabling the polymerase toselectively add the matched oligomer corresponding to the SNP identity.

The PNA-DNA chimera was able to hybridize to the DNA sequence and extendaccurately according to the corresponding match DNA monomer. The PNA-DNAchimera is able to obtain a high Match/Mismatch Ratio which wouldaccurately identify SNP-based genotyping results. Ratio forMatch/Mismatch sequence is 3.5-4 for PNA sequence while it is 65-70 orPNA-DNA chimera sequence. Thus, PNA-DNA chimera with high yield of thesequence and the ability to perform a polymerase extension step on chipdue to the DNA oligomer present provides a high-throughput, highaccuracy system for various genomics applications including, but notlimited to, SNP-based genotyping and DNA sequencing.

Example 5: Synthesis of Photoactive Molecule 1 (PM1) (dCTP-PAG1)

We modified dCTP by adding a photoacid generator (PAG1) to generatedCTP-PAG1 (i.e., “Photoactive Molecule 1”, or “PM1”). A reaction schemefor the synthesis of PM1 is shown in FIG. 14.

5-Methyl-2-nitrobenzoic acid, Thionyl Chloride, Magnesium turnings,Chlorobenzene, Carbon tetrachloride (CCl₄), Diethyl malonate, Sulfuricacid (H₂SO₄), Chloroform, Acetic acid (AcOH), Sodium carbonate (Na₂CO₃),N-bromosuccinimide, Benzoyl peroxide, Ethyl Acetate (EtOAc), Sodiumsulfate (Na₂SO₄), Hexane, Methanol, Hydrogen Chloride (HCl),1,4-dioxane, Sodium borohydride (NaBH₄), Tetrahydrofuran (THF),Triphenylphosphine (PPh₃), triphosgene, trimethylamine,N-hydroxysuccinimide, potassium hydroxide (KOH), trichloroacetic acid,and sodium bisulfite (NaHSO₃) were obtained from Sigma Aldrich. EthylAlcohol, methanol, acetonitrile and Acetone were obtained from VWR.5-Aminoallyl-2′-deoxycytidine-5′-Triphosphate was obtained from TrilinkBiotechnologies.

Step 1: 5-Methyl-2-nitrobenzoic acid (27.6 mmol) was added in smallportions to thionyl chloride (148 mmol) and the mixture was stirred atroom temperature for 12 hours. Excess thionyl chloride was removed byvacuum.

Step 2: Next, a mixture of magnesium turnings (442 mmol), 6 mL absoluteethyl alcohol, 8 mL chlorobenzene, and 0.1 mL CCl₄ were refluxed untilmost of the magnesium reacted. A solution of diethyl malonate (4.82 g)in 10 mL chlorobenzene was added followed by the addition of5-Methyl-2-nitrobenzoic acid chloride. The reaction was stirred for 1hour and 1.7 mL H₂SO₄ in 17 mL water was added. The mixture was stirredfor 20 minutes and 20 mL chloroform was added. The aqueous layer wasextracted 3 times with 10 mL chloroform and were combined, dried andevaporated.

Step 3: The residue was dissolved in 8.25 mL AcOH in 5.4 mL watercontaining 1 mL H₂SO₄ and the mixture was refluxed for 6 hours. Themixture was neutralized with aqueous Na₂CO₃, extracted 3 times with 20mL chloroform, dried and concentrated. The residue was re-crystallizedfrom 70% ethyl alcohol and isolated. This mixture is5-Methyl-2-nitroacetophenone.

Step 4: 5-Bromomethyl-2-nitroacetophenone was obtained by the mixture of5-Methyl-2-nitroacetophenone (19.6 mmol), N-bromosuccinimide (20.6 mmol)and benzoyl peroxide (0.01 meq) which were refluxed in 20 mL CCl₄ for 5hours. The reaction mixture was filtered, concentrated, recrystallizedin CCl₄ and isolated.

Step 5: The 5-Bromomethyl-2-nitroacetophenone (7.75 mmol) was dissolvedin acetone: H₂O (5:1 by volume, 50 mL). Sodium azide (11.6 mmol) wasadded, and the mixture was heated to 75° C. overnight in a flaskequipped with a reflux condenser. After acetone evaporation underreduced pressure, the aqueous phase was extracted with EtOAc, washedwith brine, and dried with Na₂SO₄, and the solvent was evaporated underreduced pressure. The mixture was purified by silica gel chromatographyusing a 10-20% gradient of EtOAc in hexane.

Step 6: The purified mixture from step 5 (6.81 mmol) was dissolved inMeOH: dioxane (3:2 by volume, 30 mL), and NaBH₄ (10 mmol) was addedslowly. After 30 min, water (50 mL) and 2 M HCl (1 mL) were added andthe suspension was extracted twice with EtOAc, washed with brine, driedover Na₂SO₄, and evaporated under reduced pressure and light protection.The resulting brown oil was purified by silica gel chromatography usinga 10-20% gradient of EtOAc in hexane.

Step 7: To a solution of compound (brown oil) from step 6, (4.54 mmol)in THF (30 mL), PPh₃ (5 mmol) and H₂O (0.5 mL) were added and themixture was heated at 60° C. for 4 h. Evaporation of the solvent underreduced pressure gave a residue that was dissolved in chloroform andpurified by silica gel chromatography with 5-15% MeOH in Chloroform togive the compound 1-(5-(Aminomethyl)-2-nitrophenyl)ethanol.

Step 8: Triphosgene (1 mmol) and triethylamine (10 mmol) were added at0° C. to the solution of the adipic acid monoethyl ester (2 mmol) indichloromethane (10 mL). Then N-hydroxysuccinimide (2 mmol) was added tothe reaction mixture. The reaction mixture was stirred for 40 min atroom temperature. After completion of the reaction, the reaction mixturewas filtered by suction filtration. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane. Thesolution was mixed with 1-(5-(Aminomethyl)-2-nitrophenyl)ethanol (3mmol) in DMF (3 mL). The solution was stirred at room temperatureovernight and evaporated in vacuum. The residue was dissolved in EtOAc,dried over Na₂SO₄, and evaporated under reduced pressure.

Step 9: To a stirring solution of the ester (1 equivalent) and methanol(5 mL), KOH (2 molar equivalents) was added at 35° C. The reaction wasallowed to continue for 1 hour and then quenched by addition of water(20 mL). The aqueous layer was acidified and the resulting acid wasextracted from the aqueous layer by EtOAc.

Step 10: A mixture of Trichloroacetic acid (1.35 mmol), 4% concentratedH₂SO₄ (2.88 μL) and 1 mL of alcohol (EtOAc containing the resultingacid) from Step 9 were combined in microwave reaction vessel andirradiated for 20 minutes, during which the acid catalyst was loadedevery 5 minutes. Once the reaction was complete, the solvent wasevaporated under pressure, and the crude residue was dissolved in ethylacetate. The organic layer was washed with NaHSO₃ and dried withanhydrous Na₂SO₄. The mixture was then purified and concentrated invacuum to give the ester product.

Step 11: Triphosgene (1 mmol) and triethylamine (10 mmol) were added at0° C. to a stirred solution of the adipic acid monoethyl ester (2 mmol)in dichloromethane (10 mL). Then N-hydroxysuccinimide (2 mmol) was addedto the reaction mixture. The reaction mixture was stirred for 40 min atroom temperature. After completion of the reaction, the reaction mixturewas filtered by suction filtration. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane. Thesolution was mixed with 1-(5-(Aminomethyl)-2-nitrophenyl)ethanol (3mmol) in DMF (3 mL). The solution was stirred at room temperatureovernight and evaporated in vacuum. The residue was dissolved in EtOAc,dried over Na₂SO₄, and evaporated under reduced pressure.

Step 12: The compound (residue) from step 11 was then dissolved in 2 mLof acetonitrile. DSC (0.75 mmol) dissolved in 3 mL of acetonitrile:ethyl acetate (5:1) solution, was then added, followed by triethylamine(0.75 mmol). 5-Aminoallyl-2′-deoxycytidine-5′-Triphosphate (1 mmol) wasthen added to this mixture. After overnight reaction, cold diluted HClwas added and the product was extracted with ethyl acetate. The solutionwas washed three times with diluted HCl, dried over anhydrous sodiumsulfate, and the solvent was removed by evaporation.

Example 6: Synthesis of Photoactive Molecule 2 (PM2) (dATP-PAG2)

We modified dATP by adding a photoacid generator (PAG2) to generatedCTP-PAG2 (i.e., “Photoactive Molecule 2” or “PM2”). A reaction schemefor the synthesis of PM2 is shown in FIG. 15. The synthesis wasperformed as follows:

N6-(6-Amino) hexyl-2′-deoxyadenosine-5′-triphosphate was obtained fromJenabiosciences. Acetic acid was obtained from Sigma Aldrich.

Steps 1 through 9 are repeated as given in Example 5.

Step 10: A mixture of Acetic acid (1.35 mmol), 4% concentrated H₂SO₄(2.88 μL) and 1 mL of alcohol (EtOAc containing the resulting acid) fromStep 9 were combined in microwave reaction vessel and irradiated for 20minutes, during which the acid catalyst was loaded every 5 minutes. Oncethe reaction was complete, the solvent was evaporated under pressure,and the crude residue was dissolved in ethyl acetate. The organic layerwas washed with NaHSO₃ and dried with anhydrous Na₂SO₄. The mixture wasthen purified and concentrated in vacuum to give the ester product.

Step 11 Triphosgene (1 mmol) and triethylamine (10 mmol) were added at0° C. to the stirred solution from Step 9 (2 mmol) in dichloromethane(10 mL). Then N-hydroxysuccinimide (2 mmol) was added to the reactionmixture. The reaction mixture was stirred for 40 min at roomtemperature. After completion of the reaction, the reaction mixture wasfiltered by suction filtration. The product was formed by the removal ofthe filtrate by rotary evaporation followed by short-path silica-gelcolumn chromatography using 20% ethyl acetate in hexane. The solutionwas mixed with 1-(5-(Aminomethyl)-2-nitrophenyl)ethanol (3 mmol) in DMF(3 mL). The solution was stirred at room temperature overnight andevaporated in vacuum. The residue was dissolved in EtOAc, dried overNa₂SO₄, and evaporated under reduced pressure.

Step 12: The resulting compound from Step 11 was dissolved in 2 mL ofacetonitrile. DSC (0.75 mmol) dissolved in 3 mL of acetonitrile: ethylacetate (5:1) solution, was then added, followed by triethylamine (0.75mmol). N6-(6-Amino) hexyl-2′-deoxyadenosine-5′-triphosphate (1 mmol) wasthen added to this mixture. After overnight reaction, cold diluted HClwas added and the product was extracted with ethyl acetate. The solutionwas washed three times with diluted HCl, dried over anhydrous sodiumsulfate, and the solvent was removed by evaporation.

Example 7: Synthesis of Photoactive Molecule 3 (PM3) (dUTP-PBG1)

We modified dUTP by adding a photobase generator (PBG1) to generatedUTP-PBG1 (i.e., “Photoactive Molecule 3” or “PM3”). A reaction schemefor the synthesis of PM3 is shown in FIG. 16. The synthesis wasperformed as follows:

3-bromo-2-iodo benzoic acid, sodium, 4-aminophenol, dimethyl sulfoxide,then tris (2-(2-methoxyethoxy) ethyl)amine, copper chloride (CuCl),1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride, Palladium(II)acetate (Pd(OAc)₂), Cesium carbonate (Cs₂CO₃), diethyl malonate,dioxane, Triazabicyclodecene, diisopropyl ether, andN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride wereobtained from Sigma Aldrich. 5-Aminoallyl-dUTP was obtained fromBiotium.

Step 1: 3-bromo-2-iodo benzoic acid (8 mmol) was dissolved in a KOHsolution (9.5 mmol). The mixture was condensed under vacuum at 100° C.and the resultant solid was heated under vacuum at 100° C. for 12 h toyield a dried potassium salt. Metallic sodium (8.35 mmol) was dissolvedin 100 mL of methanol, and 4-aminophenol (8.35 mmol) was added. Themixture was condensed and heated under vacuum at 100° C. for 12 h toyield a dried sodium salt. The sodium salt was dissolved in 15 mL ofanhydrous dimethyl sulfoxide, and then tris (2-(2-methoxyethoxy)ethyl)amine (TDA-1, 1.0 mL) was added. The mixture was stirred to reachhomogeneity at room temperature under anhydrous conditions, and thenCuCl (350 mg) and potassium salt powders of the compound were added. Theresultant mixture was stirred at 85° C. for 4 h, cooled down to roomtemperature, washed with a NaOH, filtered with diatomite, and acidifiedwith 1 N HCl to pH value at 2-3. A semisolid precipitate was collected,washed with water, and dried to yield a crude product.

Step 2: The crude product from Step 1 (7.35 mmol) was added to astirring sulfuric acid solution. The mixture was allowed to react for 30min at 90° C., and then cooled down to room temperature, diluted with200 mL of ice water, and filtered. The resultant solid was washed withwater, dried, and recrystallized from EtOAc/Methanol to yield a lightyellow solid.

Step 3: The light yellow solid from Step 2 (7.25 mmol), 1,3-bi-(2,6-diisopropylphenyl)imidazole chloride (Ipr. HCl 2% by mole),Pd(OAc)₂ (2% by mole), Cs₂CO₃ (4.7 g, 14.5 mmol), diethyl malonate (1.16g, 7.25 mmol), and 20 mL of dioxane were mixed. The mixture was allowedto react at 80° C. for 24 h, and then cooled down to room temperature,diluted with 100 mL of EtOAc, and filtered to yield a black precipitate.The filtrate was condensed and recrystallized from EtOAc/MeOH to yield alight yellow solid.

Step 4: The light yellow solid from Step 3 (0.3 mol), 350 mL ofmethanol, and a NaOH solution (0.6 mol) were mixed. The mixture washeated, stirred at 40° C., hydrolyzed completely, acidified with aceticacid to yield a white precipitate, filtered, and dried to yield a crudeproduct.

Step 5: Triphosgene (1 mmol) and triethylamine (10 mmol) were added at0° C. to a stirred solution of the adipic acid monoethyl ester (2 mmol)in dichloromethane (10 mL). Then N-hydroxysuccinimide (2 mmol) was addedto the reaction mixture. The reaction mixture was stirred for 40 min atroom temperature. After completion of the reaction, the reaction mixturewas filtered by suction filtration. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane. Thesolution was mixed with Step 5 compound (3 mmol) in DMF (3 mL). Thesolution was stirred at room temperature overnight and evaporated invacuum. The residue was dissolved in EtOAc, dried over Na₂SO₄, andevaporated under reduced pressure.

Step 6: 10 mmol of Triazabicyclodecene and 10 mmol of the compoundobtained in Step 5 were dissolved into 20 mL of acetone, and stirred atroom temperature for 30 minutes. After completion of the reaction,reaction solution was concentrated under reduced pressure, and theresulting residue was washed with diisopropyl ether, and then driedunder reduced pressure.

Step 7: To a stirring solution of the ester from Step 6 (1 equivalent)and methanol (5 mL), KOH (2 molar equivalents) was added at 35° C. Thereaction was allowed to continue for 1 hour and then quenched byaddition of water (20 mL). The aqueous layer was acidified and theresulting acid was extracted from the aqueous layer by EtOAc.

Step 8: 2 mmol of the compound from Step 7 is mixed along with EDC (8mmol) and N-hydroxysuccinimide (2 mmol) and the reaction mixture wasstirred for 40 min at room temperature. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane. Thesolution was mixed with 5-Aminoallyl-dUTP (3 mmol) in DMF (3 mL). Thesolution was stirred at room temperature overnight and evaporated invacuum. The residue was dissolved in EtOAc, dried over Na₂SO₄, andevaporated under reduced pressure.

Example 8: Synthesis of Photoactive Molecule 4 (PM4) (dGTP-PBG2)

We modified dGTP by adding a photobase generator (PBG2) to generatedUTP-PBG2 (i.e., “Photoactive Molecule 4” or “PM4”). A reaction schemefor the synthesis of PM4 is shown in FIG. 17. The synthesis wasperformed as follows:

1,8-Diazabicyclo[5.4.0]undec-7-ene was obtained from Sigma Aldrich.7-Deaza-7-Propargylamino-2′-deoxyguanosine-5′-Triphosphate was obtainedfrom Trilink Biotechnologies.

Steps 1 through 5 are repeated as given in Example 7.

Step 6: 10 mmol of 1,8-Diazabicyclo[5.4.0]undec-7-ene and 10 mmol of thecompound obtained in Step 5 were dissolved into 20 mL of acetone, andstirred at room temperature for 30 minutes. After completion of thereaction, reaction solution was concentrated under reduced pressure, andthe resulting residue was washed with diisopropyl ether, and then driedunder reduced pressure.

Step 7: To a stirring solution of the ester from Step 6 (1 equivalent)and methanol (5 mL), KOH (2 molar equivalents) was added at 35 C. Thereaction was allowed to continue for 1 hour and then quenched byaddition of water (20 mL). The aqueous layer was acidified and theresulting acid was extracted from the aqueous layer by EtOAc.

Step 8: 2 mmol of the compound from Step 7 is mixed along with EDC (8mmol) and N-hydroxysuccinimide (2 mmol) and the reaction mixture wasstirred for 40 min at room temperature. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane. Thesolution was mixed with7-Deaza-7-Propargylamino-2′-deoxyguanosine-5′-Triphosphate (3 mmol) inDMF (3 mL). The solution was stirred at room temperature overnight andevaporated in vacuum. The residue was dissolved in EtOAc, dried overNa₂SO₄, and evaporated under reduced pressure.

Example 9: Testing of Performance of Photoactive Molecules

Photoactive molecules PM1-PM4 were tested for their performance inPolymerase Chain Reactions (PCR) and specifically genotyping applicationon the methylenetetrahydrofolate reductase (MTHFR) gene. The nucleotidepolymorphism 677 C>T (rs1801133) is located within the region coding forthe catalytic domain of MTHFR and results in an amino acid substitutionfrom an alanine to a valine at codon position 222 (exon 4).

PNA sequences were synthesized on a Silicon wafer containing amicroarray of sequences to be used as ‘adapters’ for allele specificprimer extension reactions. Genomic DNA samples were obtained in-housefrom the Vibrant Genomics Labs and the mutations of 677C>T were knownthrough ACGT, Inc. sequencing.

To amplify the region in the genomic DNA, PCR was performed in a 20 μLreaction volume as follows: 50 ng of the human genomic DNA was added toa PCR amplification reagent mixture comprising: 2.5 units of TitaniumTaq DNA polymerase (Clontech), 200 μM each dNTP (dATP, dTTP, dGTP, dCTP)(Thermofisher Scientific), 10× PCR Buffer with 1.5 mM MgCI2(Thermofisher Scientific), 20 pmol sense primer(5′-CCTATTGGCAGGTTACCCCA-3′) (SEQ ID NO: 22) (IDT), and 20 pmolantisense primer (5′-GGCAAGTGATGCCCATGTCG-3′) (SEQ ID NO: 23) (IDT). ThePCR reaction mixture was then pre-denatured at 94° C. for 5 minutes,followed by 35 cycles of amplification, each cycle comprising 1)denaturation at 94° C. for 30 seconds, 2) annealing at 54° C. for 30seconds, and 3) extension at 72° C. for 45 seconds. A final extensionstep at 72° C. for 10 minutes was performed.

Primer extension mixes were prepared using extension primers specific toalleles. In this particular assay, ATGGCTAG-GAAGGTGTCTGCGGGAGC (SEQ IDNO: 1) and CGCGATTG-GAAGGTGTCTGCGGGAGT (SEQ ID NO: 2) (IDT) were usedand represented as Adapter-Primer with the former being the primerspecific to Wild-Type allele (‘C’) and the latter being the primerspecific to the Mutant-Type allele (‘T’) of the MTHFR gene. Themicroarray contains sequences complimentary to the above adapters todetect the performance of individual primers.

To test the performance of individual photoactive molecules, 4 differentprimer extension mixes were used. Primer Extension Mix 1 (PEM1)comprised of 20 μL reaction volume with 2.5 units of Titanium Taq DNApolymerase, 50 μM each dNTP (dATP, dTTP, dGTP, dCTP) and 10 μM of PM1,10×PCR Buffer with 1.5 mM MgCl₂ (Thermofisher Scientific), and 20 pmolprimer extension mix containing primers for both wild-type (SEQ IDNO: 1) and mutant-type (SEQ ID NO: 2) allele specific primers.

Primer Extension Mix 2 (PEM2) comprised of 20 μL reaction volume with2.5 units of Titanium Taq DNA polymerase, 50 μM each dNTP (dATP, dTTP,dGTP, dCTP) and 10 μM of PM2, 10×PCR Buffer with 1.5 mM MgCl₂(Thermofisher Scientific), 20 pmol primer extension mix containingprimers for both wild-type (SEQ ID NO: 1) and mutant-type (SEQ ID NO: 2)allele specific primers.

Primer Extension Mix 3 (PEM3) comprised of 20 μL reaction volume with2.5 units of Titanium Taq DNA polymerase, 50 μM each dNTP (dATP, dTTP,dGTP, dCTP) and 10 μM of PM3, 10×PCR Buffer with 1.5 mM MgCl₂(Thermofisher Scientific), 20 pmol primer extension mix containingprimers for both wild-type (SEQ ID NO: 1) and mutant-type (SEQ ID NO: 2)allele specific primers.

Primer Extension Mix 4 (PEM4) comprised of 20 μL reaction volume with2.5 units of Titanium Taq DNA polymerase, 50 μM each dNTP (dATP, dTTP,dGTP, dCTP) and 10 μM of PM4, 10×PCR Buffer with 1.5 mM MgCl₂(Thermofisher Scientific), 20 pmol primer extension mix containingprimers for both wild-type (SEQ ID NO: 1) and mutant-type (SEQ ID NO: 2)allele specific primers.

Primer Extension reaction was done by adding 20 μL of primer extensionmix to the amplified reaction mix and performed 40 cycles of PCR, with apre-denaturation at 94° C. for 5 minutes and each cycle comprising ofdenaturation at 94° C. for 30 seconds, annealing/extension at 54° C. for30 seconds, were carried out, followed by a final post-denaturation at94° C. for 5 minutes.

After the completion of primer extension reaction, 60 μl ofhybridization buffer (1× PBS buffer with 1% fish gelatin and 0.1 mg/mLsalmon sperm DNA) was added and the pillar plate was kept in ahybridization chamber at 45° C. for 2 hours. This was followed by 2 washsteps of PBS with the first wash being at 39° C. for 5 minutes followedby a wash at room temperature for 10 minutes. The chips were then rinsedwith DI water and dried under a stream of nitrogen.

The chips were then exposed using 365 nm bulb strata-linker for 15minutes and mixed with 30 μl of DI water. Lastly, the pH of the solutionwas read using an ISFET pH sensor (Sentron). The results obtained areshown in FIGS. 18, 19A, 19B, 19C and 19D.

Example 10: Synthesis of Photoactive Molecule 5 (PM5) (ddCTP-PAG1)

Photoactive molecule PM5 is synthesized as given in Example 5. dCTP isreplaced by ddCTP in this molecule. The structure of PM5 is shown inFIG. 20.

Example 11: Synthesis of Photoactive Molecule 6 (PM6) (ddATP-PAG2)

Photoactive molecule PM6 is synthesized as given in Example 6. dATP isreplaced by ddATP in this molecule. The structure of PM6 is shown inFIG. 21.

Example 12: Synthesis of Photoactive Molecule 7 (PM7) (ddUTP-PBG1)

Photoactive molecule PM7 is synthesized as given in Example 7. dUTP isreplaced by ddUTP in this molecule. The structure of PM7 is shown inFIG. 22.

Example 13: Synthesis of Photoactive Molecule 8 (PM8) (ddGTP-PBG2)

Photoactive molecule PM8 is synthesized as given in Example 8. dGTP isreplaced by ddGTP in this molecule. The structure of PM8 is shown inFIG. 23.

Example 14: Testing the Performance of Photoactive Molecules (HM 5-8)

Photoactive molecules were tested for their performance in PolymeraseChain Reactions (PCR) and specifically genotyping application on themethylenetetrahydrofolate reductase (MTHFR) and 9p21 gene. Thenucleotide polymorphism 677 C>T (rs1801133) is located within the regioncoding for the catalytic domain of MTHFR and results in an amino acidsubstitution from an alanine to a valine at codon position 222 (exon 4).The polymorphism rs10757274 (A;G) is a SNP located in chromosomal region9p21.

Genotyping of MTHFR and 9p21 regions, with the well-known mutations,rs1801133 and rs10757274, were tested using 20 DNA samples. The DNAsamples had a known genotyping result which were determined usingReal-Time PCR. 34-mer length PNA-DNA chimera primer sequences5′-CTGAAGCACTTGAAGGAGAAGGTGTCTGCGG(GAG)-3′ (SEQ ID NO: 16) for thers1801133 mutation and 5′-CTCC CCCGTGGGTCAAATCTAAGC TGAGTG(TTG)-3′ (SEQID NO: 24) for the rs10757274 mutation were synthesized according to themethods given above.

DNA were extracted from the samples (buccal swabs) using methods knownto one skilled in the art. A standard PCR reaction using forward andreverse primers was performed on the extracted DNA samples.Hybridization and polymerase extension on each of chips were performedas follows. The PCR product was mixed in 1×DNA Polymerase Buffer(Clontech), 20 nmol of MgCl₂, 1 unit Titanium Taq DNA Polymerase(Clontech), and all 4 photoactive molecules (PM5, PM6, PM7, PM8) (each20 pmoles). Hybridization is done in a hybridization chamber at 55° C.for 30 minutes followed by washing the chips in 0.1× Ssarc buffer 40° C.for 5 minutes twice followed by rinsing in DI Water. The chips were thenexposed using 365 nm bulb strata-linker for 15 minutes and mixed with 30μl of DI water. Lastly, the pH of the solution was read using an ISFETpH sensor (Sentron). The results obtained are shown in FIGS. 24A and24B.

Example 15: Synthesis of Anti-p53 Bound to PBG1

3-bromo-2-iodo benzoic acid, sodium, 4-aminophenol, dimethyl sulfoxide,then tris (2-(2-methoxyethoxy) ethyl)amine, copper chloride (CuCl),1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride, Palladium(II)acetate (Pd(OAc)2), Cesium carbonate (Cs2CO3), diethyl malonate,dioxane, Triazabicyclodecene, diisopropyl ether, andN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride wereobtained from Sigma Aldrich.

Step 1: 3-bromo-2-iodo benzoic acid (8 mmol) was dissolved in a KOHsolution (9.5 mmol). The mixture was condensed under vacuum at 100° C.and the resultant solid was heated under vacuum at 100° C. for 12 h toyield a dried potassium salt. Metallic sodium (8.35 mmol) was dissolvedin 100 mL of methanol, and 4-aminophenol (8.35 mmol) was added. Themixture was condensed and heated under vacuum at 100° C. for 12 h toyield a dried sodium salt. The sodium salt was dissolved in 15 mL ofanhydrous dimethyl sulfoxide, and then tris (2-(2-methoxyethoxy)ethyl)amine (TDA-1, 1.0 mL) was added. The mixture was stirred to reachhomogeneity at room temperature under anhydrous conditions, and thenCuCl (350 mg) and potassium salt powders of the compound were added. Theresultant mixture was stirred at 85° C. for 4 h, cooled down to roomtemperature, washed with a NaOH, filtered with diatomite, and acidifiedwith 1 N HCl to pH value at 2-3. A semisolid precipitate was collected,washed with water, and dried to yield a crude product.

Step 2: The crude product from Step 1 (7.35 mmol) was added to astirring sulfuric acid solution. The mixture was allowed to react for 30min at 90° C., and then cooled down to room temperature, diluted with200 mL of ice water, and filtered. The resultant solid was washed withwater, dried, and recrystallized from EtOAc/Methanol to yield a lightyellow solid.

Step 3: The compound from Step 2 (7.25 mmol), 1,3-bi-(2,6-diisopropylphenyl)imidazole chloride (Ipr. HCl 2% by mole),Pd(OAc)2 (2% by mole), Cs2CO3 (4.7 g, 14.5 mmol), diethyl malonate (1.16g, 7.25 mmol), and 20 mL of dioxane were mixed. The mixture was allowedto react at 80° C. for 24 h, and then cooled down to room temperature,diluted with 100 mL of EtOAc, and filtered to yield a black precipitate.The filtrate was condensed and recrystallized from EtOAc/MeOH to yield alight yellow solid.

Step 4: The compound from Step 3 (0.3 mol), 350 mL of methanol, and aNaOH solution (0.6 mol) were mixed. The mixture was heated, stirred at40° C., hydrolyzed completely, acidified with acetic acid to yield awhite precipitate, filtered, and dried to yield a crude product.

Step 5: Triphosgene (1 mmol) and triethylamine (10 mmol) were added atOC to a stirred solution of the adipic acid monoethyl ester (2 mmol) indichloromethane (10 ml). Then N-hydroxysuccinimide (2 mmol) was added tothe reaction mixture. The reaction mixture was stirred for 40 min atroom temperature. After completion of the reaction, the reaction mixturewas filtered by suction filtration. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane. Thesolution was mixed with Step 5 compound (3 mmol) in DMF (3 ml). Thesolution was stirred at room temperature overnight and evaporated invacuum. The residue was dissolved in EtOAc, dried over Na2SO4, andevaporated under reduced pressure.

Step 6: 10 mmol of Triazabicyclodecene and 10 mmol of the compoundobtained in Step 5 were dissolved into 20 mL of acetone, and stirred atroom temperature for 30 minutes. After completion of the reaction,reaction solution was concentrated under reduced pressure, and theresulting residue was washed with diisopropyl ether, and then driedunder reduced pressure.

Step 7: To a stirring solution of the ester from Step 6 (1 equivalent)and methanol (5 mL), KOH (2 molar equivalents) was added at 35 C. Thereaction was allowed to continue for 1 hour and then quenched byaddition of water (20 mL). The aqueous layer was acidified and theresulting acid was extracted from the aqueous layer by EtOAc.

Step 8: 2 mmol of the compound from Step 7 is mixed along with EDC (8mmol) and N-hydroxysuccinimide (2 mmol) and the reaction mixture wasstirred for 40 min at room temperature. The product was formed by theremoval of the filtrate by rotary evaporation followed by short-pathsilica-gel column chromatography using 20% ethyl acetate in hexane.

Step 9: The solution was mixed with Anti-p53 antibody (3 mmol) in water(3 ml). The reaction was carried out at room temperature overnight andpurified using a spin column.

Example 16: Testing Detection of the Photoactive Molecule Anti-p53 Boundto PBG1

Wafers with COOH are prepared as described previously. 4 weight % for1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 2 weight % forN-Hydroxysuccinimide (NHS) were added in 94 weight % DI water asreagents to activate the COOH substrate. Wafer substrate is activated byspinning the wafer with this reagent wash solution and then washed awaywith water.

All Fmoc based amino acids required for p53 validation on the wafer areobtained from Anaspec. The amino acids obtained are Leucine (Leu),Lysine (Lys), Tryptophan (Trp), Aspartic Acid (Asp), Serine (Ser),Phenylalanine (Phe), Threonine (Thr), Glutamic acid (Glu), Glutamine(Gln), Arginine (Arg) and Histidine (His). Ethanolamine is obtained fromSigma Aldrich.

Validation of the wafer by p53 coupling is done by coupling 2sequences—natural and mutated sequence for p53 antibody. The naturalsequence grown is Leu-Lys-Trp-Leu-Asp-Ser-Phe-Thr-Glu-Gln (SEQ ID NO:25) and mutated sequence grown isLeu-Lys-Trp-Leu-Arg-His-Phe-Thr-Glu-Gln (SEQ ID NO: 26).

Amino acid coupling is done as follows—A Photobase coupling solutioncontaining a polymer, amino acid and photobase is spin coated onto awafer at 3000 rpm and baked at 65c for 1 min. in a hot plate. Now thewafer is exposed using a reticle at 80 mj/cm2. and then hard baked at85c for 90 sec in a hot plate. Fmoc is deprotected only in the regionwhere it is exposed and the amino acid is coupled simultaneously.

1M Ethanolamine is used for capping the activated COOH which have notbeen coupled. This is done by spin coating a mixture of polymer+1MEthanolamine+DI Water and baked at 65 C for 90 secs. Wafer is thenstripped with Acetone and IPA and the same process is repeated forcoupling the next layer of amino acid.

Natural sequence starts with coupling Leu and is completed by couplingGln. This sequence is grown on the wafer using a specific mask. Anotherindependent mask is used to grow the mutated sequence similarly. Thewafer is diced into chips and bioassay is done to verify p53 sequenceassay sensitivity comparing 2 methods of detection. The first method isusing fluorescence and the second method is using pH sensing.

Anti-p53 Antibody and Atto 488 Goat ani-mouse IgG are obtained fromABCAM. TBS Buffer, PBST Buffer and BSA are obtained from VWR. Thebioassay process is done as follows:

Fluorescence detection: The chip is washed with methanol for 5 minsfollowed by washing with TBS Buffer for 5 mins. Primary antibodycontaining PBST+1% BSA+Anti-p53 Antibody is incubation is done on thechip at 37 C for 1 hr. The chip is washed with PBST for 5 mins thrice.This is followed by secondary antibody incubation at 37 C for 1 hr.Secondary antibody contains PBST+1% BSA+Goat Anti-mouse IgG. The chip iswashed with PBST for 5 mins thrice. This is followed by DI water washingfor 5 mins twice. The chips are dried and scanned under a 488 nm laserscanner.

pH detection: The chip is washed with methanol for 5 mins followed bywashing with TBS Buffer for 5 mins. Primary antibody containing PBST+1%BSA+Anti-p53 Antibody photoactive molecule (PBG1-p53) is incubation isdone on the chip at 37 C for 1 hr. The chip is washed with PBST for 5mins thrice. This is followed by DI water washing for 5 mins twice. Thechips were then dried and exposed using 365 nm bulb strata-linker for 15minutes and mixed with 30 ul of DI water and the pH of the solution wasread using an ISFET pH sensor (Sentron).

The results are shown in Table 6 below:

TABLE 6 Results of ionic vs. fluorescent detection of antibody bindingDetection Method: Fluorescence Detection Method: Fluorescence p53 De-p53 De- ID Antibody Signal tected ID Antibody pH tected On Naturalsequence synthesized S1 1 ng/ml 65535 Y S1 1 ng/ml 12.3 Y S2 100 pg/ml12478 Y S2 100 pg/ml 11.2 Y S3 10 pg/ml 2645 Y S3 10 pg/ml 9.9 Y S4 1pg/ml 856 N S4 1 pg/ml 8.4 Y S5 0 pg/ml 800 N S5 0 pg/ml 7.23 N OnMutant sequence synthesized S1 1 ng/ml 756 N S1 1 ng/ml 7.31 N S2 100pg/ml 745 N S2 100 pg/ml 7.38 N S3 10 pg/ml 700 N S3 10 pg/ml 7.47 N S41 pg/ml 536 N S4 1 pg/ml 7.45 N S5 0 pg/ml 275 N S5 0 pg/ml 7.38 N

The results indicate that the photoactive molecules concept can beincorporated into protein/antibody detection and has a highersensitivity of <1 pg/ml compared to 10 pg/ml (for fluorescencedetection). This may be attributed to that the fact the fluorescencedetection includes excitation which leads to higher noise compared to pHsensing. The pH sensing model can be utilized for protein and antibodydetection assays.

Example 17: Sequencing

Photoactive sequencing molecules PM 9 (NPPOC-3′-dCTP-PAG1), PM10(NPPOC-3′-dATP-PAG2), PM11 (NPPOC-3′-dUTP-PBG1) and PM 12(NPPOC-3′-dGTP-PBG2) are synthesized as given in Examples 5, 6, 7 and 8by replacing the dNTP's with 3′-NPPOC blocked nucleotides. The NPPOCblocking protects the nucleotide extension until the group is removed(i.e., is a removable blocker). Photoactive sequencing moleculesPM9-PM12 are tested for their performance of sequencing on themethylenetetrahydrofolate reductase (MTHFR) gene.

A PNA-DNA chimera sequence 5′-ATGCACCGACATGGGC-3′ (SEQ ID NO: 18) issynthesized on a Silicon wafer with ISFET wells according to the methodspreviously described. Genomic DNA samples are obtained in-house from theVibrant Genomics Labs and sequenced using a commercial synthesizer todetermine the sequence of the region of interest. The sequence to besynthesized is 5′-ATGCACCGACATGGGC ATCACTTG-3′ (SEQ ID NO: 19), with thesequencing by synthesis component in bold.

A standard PCR reaction using forward and reverse primers is performedon the extracted DNA samples to amplify the MTHFR gene. Hybridization ofthe amplification product to the PNA-DNA probes and polymerase extensionfor sequencing by synthesis is performed. The PCR product is mixed in1×DNA Polymerase Buffer (Clontech), 20 nmol of MgCl₂, 1 unit TitaniumTaq DNA Polymerase (Clontech), and all 4 photoactive molecules (PM9,PM10, PM11, PM12) (each 20 pmoles).

Hybridization is done in a hybridization chamber at 55° C. for 30minutes followed by washing the chips in 0.1× Ssarc buffer 40° C. for 5minutes twice followed by rinsing in DI Water.

The chips are then exposed using 365 nm bulb strata-linker for 15minutes and mixed with 30 μl of DI water. The pH of the solution is readusing an ISFET pH sensor (Sentron).

The exposure to 365 nm light also simultaneously unblocks the NPPOCprotection on the 3′ end to enable continuation of the polymerizationreaction and sequencing by synthesis. This hybridization and extensioncycle are repeated multiple times to generate and detect the sequence ofthe region of interest. The results anticipated are shown in Table 7:

TABLE 7 Expected results of sequencing pH Detected Cycle Reading PM Callnucleotide Cycle 1 3.84 PM10 A Cycle 2 11.2 PM11 T Cycle 3 2.68 PM9 CCycle 4 3.98 PM10 A Cycle 5 2.66 PM9 C Cycle 6 11.02 PM11 T Cycle 7 11.1PM11 T Cycle 8 10.15 PM12 G

As shown, the detected nucleotide sequence will be 5′-ATCACTTG-3′ (SEQID NO: 20), corresponding with the amplification product from the MTHFRamplicon sequence 5′-CAAGTGAT-3′ (SEQ ID NO: 21)

Thus, the photoactive sequencing molecules described herein can beutilized to determine the sequence of a polynucleotide using reversibleterminator and a photoactive compound attached to the mononucleotides.

OTHER EMBODIMENTS

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

What is claimed is:
 1. A method of determining a sequence of a targetpolynucleotide, comprising: providing an array comprising a plurality ofwells, wherein said wells comprise a target polynucleotide to besequenced bound to a surface of said well, and wherein said plurality ofwells each comprise a sensor for detecting an electronic signal fromsaid wells; performing a sequencing reaction comprising performing atleast one cycle, each cycle comprising: contacting said wells with asolution comprising reagents for performing a polymerase extensionreaction, said solution comprising a modified nucleotide comprising aphotoactive group and a removable blocking group; exposing said well toconditions to promote incorporation of one of said modified nucleotidesat the 3′ end of a primer or probe hybridized to said singlepolynucleotide; washing said well to remove unbound modifiednucleotides; exposing said well to a wavelength of light to induce saidphotoactive group to generate and acid or a base, thereby generating adetectable change in ion concentration; detecting the change in ionconcentration with said sensor; and if another cycle of the sequencingreaction is to be performed, removing said removable blocking group fromsaid incorporated nucleotide.
 2. The method of claim 1, wherein saidelectronic signal is specific to the identity of the base of themodified nucleotide added to the primer at each cycle.
 3. The method ofclaim 1, wherein said electronic signal represents the pH of a solutionin said well.
 4. The method of claim 1, wherein said electronic signalis analyzed to determine a sequence of the target polynucleotide.
 5. Themethod of claim 1, wherein said sensor is an ion-sensitive field effecttransistor.
 6. The method of claim 1, wherein said modified nucleotidescomprise a nucleotide according to Formula I:

wherein n is from 0-3; X is selected from the group consisting of: H,OPg, and a photoactive group, where Pg is a protecting group; A is NHwhen

and A is N when

E is O when

and E is NHZ when

and each Z is independently selected from the group consisting of: H,Me, and a photoactive group; wherein at least one of said Z or X is saidphotoactive group.
 7. The method of claim 1, wherein said modifiednucleotides comprise a nucleotide according to Formula II:

wherein n is from 0-3; X is selected from the group consisting of: H,OPg, and a photoactive group, where Pg is a protecting group; A is NHwhen

and A is N when

E is O when

and E is NHZ when

and each Z is independently selected from the group consisting of: H,Me, and a photoactive group; wherein at least one of said Z or X is saidphotoactive group.
 8. The method of claim 1, wherein said photoactivegroup is a photoacid generator or a photobase generator.
 9. The methodof claim 1, wherein said modified nucleotides comprise a nucleotideselected from the group consisting of: PM1, PM2, PM3, PM4, PM5, PM6,PM7, and PM8.
 10. The method of claim 1, wherein said removable blockinggroup is a reversible terminator.
 11. The method of claim 1, wherein thephotoactive group is photocleavable.
 12. The method of claim 1, whereinthe photoactive group is a photoacid or photobase generator.
 13. Themethod of claim 1, wherein said set of modified nucleotides comprisesonly one of the group consisting of: nucleotides comprising adenine,nucleotides comprising guanine, nucleotides comprising thymine, ornucleotides comprising cytosine.
 14. The method of claim 1, wherein saidset of modified nucleotides comprises nucleotides comprising adenine,guanine, cytosine, and thymine or uracil.
 15. The method of claim 1,wherein said solution comprises a plurality of random primers.
 16. Themethod of claim 1, wherein said reagents for performing a polymeraseextension reaction comprise a primer capable of hybridizing to saidsingle polynucleotide.
 17. The method of claim 1, wherein, if anothercycle is to be performed, the method further includes neutralizing thesolution in the wells.
 18. The method of claim 1, wherein said pluralityof wells each comprise only a single target polynucleotide.
 19. Themethod of claim 1, wherein said plurality of wells each comprise aclonal population of a target polynucleotide.
 20. The method of claim 1,comprising performing 2 or more of said cycles, 5 or more of saidcycles, 10 or more of said cycles, 20 or more of said cycles, or 50 ormore cycles of said cycles.
 21. A method of detecting a sequenceidentity of a target polynucleotide, comprising: providing a substratean immobilized target polynucleotide hybridized to a primer or probe;contacting said immobilized target polynucleotide with a solutioncomprising reagents for performing a polymerase extension reaction, saidsolution comprising a set of modified nucleotides comprising aphotoactive group and a blocking group; exposing said substrate toconditions to promote incorporation of one of said modified nucleotidesat the 3′ end of said primer or probe; washing said substrate to removeunbound modified nucleotides; exposing said immobilized targetpolynucleotide to a wavelength of light to induce said photoactive groupto generate an acid or a base, thereby generating a detectable change inion concentration in a solution surrounding said immobilized targetpolynucleotide if said modified nucleotide is incorporated into saidtarget polynucleotide; detecting said change in ion concentration; anddetermining a sequence identity of said target polynucleotide from saiddetected change in ion concentration.
 22. A method for detecting atarget biomolecule, comprising: providing probe capable of bindingspecifically to a target biomolecule, wherein said probe is bound to aphotoacid generator or a photobase generator; contacting a samplesuspected of comprising said target biomolecule with said probe;removing unbound probes from said sample; exposing said sample to anwavelength of light capable of activating said photoacid generator orsaid photobase generator, such that said probe, if bound to said targetbiomolecule, releases an acid or a base upon exposure to said wavelengthof light; and detecting a concentration of ions in the sample, therebyidentifying the presence or absence of said target analyte based on achange of said concentration of ions.
 23. A probe capable of bindingspecifically to a target biomolecule, wherein said probe is bound to aphotoactive group.
 24. The probe of claim 23, wherein said probe is anucleotide bound to a photoacid generator or a photobase generator.